Methods and apparatus for single molecule sequencing using energy transfer detection

ABSTRACT

Provided herein are systems and methods for nucleotide incorporation reactions. The systems comprise polymerases having altered nucleotide incorporation kinetics and are linked to an energy transfer donor moiety, and nucleotide molecules linked with at least one energy transfer acceptor moiety. The donor and acceptor moieties undergo energy transfer when the polymerase and nucleotide are proximal to each other during nucleotide binding and/or nucleotide incorporation. As the donor and acceptor moieties undergo energy transfer, they generate an energy transfer signal which can be associated with nucleotide binding or incorporation. Detecting a time sequence of the generated signals, or the change in the signals, can be used to determine the order of the incorporated nucleotides, and can therefore be used to deduce the sequence of the target molecule.

This application is a divisional of U.S. application Ser. No. 15/621,341filed on Jun. 13, 2017, now allowed, which is a divisional of U.S.application Ser. No. 14/584,829 filed on Dec. 29, 2014, now U.S. Pat.No. 9,695,471, which is a divisional of U.S. application Ser. No.13/562,159, filed on Jul. 30, 2012, now U.S. Pat. No. 8,999,674, whichis a continuation of U.S. application Ser. No. 12/748,168, filed on Mar.26, 2010, now abandoned, which claims the filing date benefit of U.S.Provisional Application Ser. No. 61/307,356, filed on Feb. 23, 2010;61/299,917, filed on Jan. 29, 2010; 61/299,919, filed on Jan. 29, 2010;61/293,616, filed on Jan. 8, 2010; 61/293,618, filed on Jan. 8, 2010;61/289,388; filed on Dec. 22, 2009; 61/263,974, filed on Nov. 24, 2009;61/245,457, filed on Sep. 24, 2009; 61/242,771, filed on Sep. 15, 2009;61/184,770, filed on Jun. 5, 2009; and 61/164,324, filed on Mar. 27,2009. The contents of each foregoing patent applications areincorporated by reference in their entirety.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 14, 2010, is namedLT00019.txt and is 84,083 bytes in size.

FIELD

The disclosed embodiments are related generally to single moleculesequencing. More specifically, the disclosed embodiments relate to anenergy transfer system which permits detection and monitoring ofnucleotide polymerization.

BACKGROUND

Obtaining nucleic acid sequence information is an important startingpoint for medical and academic research endeavors. The sequenceinformation facilitates medical studies of active disease, geneticdisease predispositions, and assists in rational design of drugstargeting specific diseases. Sequence information is also the basis forgenomic and evolutionary studies, and many genetic engineeringapplications. Reliable sequence information is critical for paternitytests, criminal investigations, and forensic studies.

Nucleic acid sequence information is typically obtained using chaintermination and size separation procedures, such as those described bySanger, et al., (1977 Proc. Nat. Acad. Sci. USA 74:5463-5467). Prior togel separation, the nucleic acid target molecules of interest arecloned, amplified, and isolated. Then the sequencing reactions areconducted in four separate reaction vessels, one for each nucleotide: A,G, C and T. These sequencing methods are adequate for read lengths of500-10000 nucleotides. However, they are time-consuming and requirerelatively large amounts of target molecules. Additionally, thesemethods can be expensive, as they require reagents for four reactionvessels. And the amplification steps are error-prone which canjeopardize acquiring reliable sequence information. Furthermore, thesemethods suffer from sequence-dependent artifacts including bandcompression during size separation.

The technological advances in automated sequencing machines,fluorescently-labeled nucleotides, and detector systems, have improvedthe read lengths, and permit massively parallel sequencing runs for highthroughput methods. But these procedures are still inadequate for largeprojects, like sequencing the human genome. The human genome containsapproximately three billion bases of DNA sequence. Procedures that cansequence and analyze the human genome (or the genome of any organism) ina relatively short time span and at a reduced cost will make it feasibleto deliver genomic information as part of a healthcare program which canprevent, diagnose, and treat disease.

The energy transfer system provided herein overcomes many problemsassociated with current nucleotide incorporation procedures. The energytransfer system requires minute amounts of target molecule with noamplification steps, there is no need to perform four separatenucleotide incorporation reactions, and the reactions are not sizeseparated or loaded on a gel. The energy transfer system is a singlemolecule sequencing system which facilitates rapid, accurate, andreal-time sequencing of long nucleic acid fragments.

SUMMARY

In one embodiment, the disclosed relates to methods for generating anenergy transfer signal, comprising: contacting (i) a polymerase havingaltered nucleotide incorporation kinetics and linked to an energytransfer donor moiety with (ii) a nucleic acid molecule and with (iii)at least one type of a nucleotide having an energy transfer acceptormoiety, so as to incorporate the nucleotide into the nucleic acidmolecule thereby locating the polymerase and nucleotide in closeproximity with each other to generate the energy transfer signal.

In another embodiment, the disclosed relates to methods for generatingan energy transfer signal comprising the steps of: contacting (i) apolymerase having altered nucleotide incorporation kinetics and linkedto an energy transfer donor moiety with (ii) a nucleic acid molecule andwith (iii) at least one type of a hexaphosphate nucleotide having anenergy transfer acceptor moiety, so as to incorporate the nucleotideinto the nucleic acid molecule thereby locating the polymerase andnucleotide in close proximity with each other to generate the energytransfer signal.

In another embodiment, the disclosed relates to methods for generatingan energy transfer signal comprising the steps of: contacting (i) apolymerase having altered nucleotide incorporation kinetics and linkedto an energy transfer donor moiety with (ii) a target nucleic acidmolecule which is base-paired with a polymerization initiation sitehaving a terminal 3′ OH group and with (iii) at least one type of anucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide onto the terminal 3′ OH group therebylocating the polymerase and nucleotide in close proximity with eachother to generate the energy transfer signal.

In another embodiment, the disclosed relates to methods for generatingan energy transfer signal comprising the steps of: contacting (i) apolymerase having altered nucleotide incorporation kinetics and linkedto an energy transfer donor moiety with (ii) a target nucleic acidmolecule which is base-paired with a polymerization initiation sitehaving a terminal 3′ OH group and with (iii) at least one type of ahexaphosphate nucleotide having an energy transfer acceptor moiety, soas to incorporate the nucleotide onto the terminal 3′ OH group therebylocating the polymerase and nucleotide in close proximity with eachother to generate the energy transfer signal.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates non-limiting examples of the general structures ofnucleotides linked with at least one energy transfer acceptor moiety.

FIG. 2 depicts one embodiment showing an immobilized targetmolecule/primer duplex to re-sequence the same target molecule, in adirection away from the solid surface, using the reagent exchangemethods.

FIG. 3 depicts another embodiment showing an immobilized targetmolecule/primer duplex to re-sequence the same target molecule, in adirection away from solid surface, using the reagent exchange methods.

FIG. 4 depicts another embodiment showing an immobilized, self-primedtarget molecule to re-sequence the same target molecule, in a directionaway from the solid surface, using the reagent exchange methods.

FIG. 5A depicts one embodiment showing an immobilized targetmolecule/primer duplex to synthesize an extension product, where thesame extension product is re-sequenced in a direction towards the solidsurface, using the reagent exchange methods.

FIG. 5B depicts one embodiment showing an immobilized targetmolecule/primer duplex to synthesize an extension product, where thesame extension product is re-sequenced in a direction towards the solidsurface, using the reagent exchange methods.

FIG. 6A depicts another embodiment showing an immobilized targetmolecule/primer duplex to synthesize an extension product, where thesame extension product is re-sequenced in a direction towards the solidsurface, using the reagent exchange methods.

FIG. 6B depicts another embodiment showing an immobilized targetmolecule/primer duplex to synthesize an extension product, where thesame extension product is re-sequenced in a direction towards the solidsurface, using the reagent exchange methods.

FIG. 7 depicts one embodiment showing an immobilized circular targetnucleic acid molecule and a primer for rolling circle replication tore-sequence the same target molecule multiple times.

FIG. 8 depicts one embodiment showing an immobilized double-strandedtarget nucleic acid molecule, which is ligated at both ends withadaptors, for rolling circle replication to re-sequence the same targetmolecule multiple times.

FIG. 9A is a graph showing the binding curve of labeled oligonucleotidesbinding to UDG-ugi-C8 nanoparticle-HP1-phi29 polymerase conjugates. Inthis graph, mP is millipolarization units.

FIG. 9B is a mathematical equation that describes the binding curveshown in FIG. 9A, and was used to calculate the number of active phi29polymerases per nanoparticle. The concentration of the nanoparticlescontained in the polymerase-nanoparticle conjugates is known, but thenumber of phi29 polymerases (X) per nanoparticle is unknown.

FIG. 9C is a table that lists the number of active phi29 polymerases perpolymerase-nanoparticle conjugate, obtained by applying the equationshown in FIG. 9B.

FIG. 10A is a graph showing the binding curve of labeledoligonucleotides binding to UDG-ugi-C8 nanoparticle-HP1-B103 polymeraseconjugates. In this graph, mP is millipolarization units.

FIG. 10B is a mathematical equation that describes the binding curveshown in FIG. 10A, and was used to calculate the number of active B103polymerases per nanoparticle. The concentration of the nanoparticlescontained in the polymerase-nanoparticle conjugates is known, but thenumber of B103 polymerases (X) per nanoparticle is unknown.

FIG. 10C is a table that lists the number of active B103 polymerases perpolymerase-nanoparticle conjugate, obtained by applying the equationshown in FIG. 10B.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is explicitly orimplicitly set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A LaboratoryManual, Third Edition; Ausubel, F. M., et al., eds., 2002, ShortProtocols In Molecular Biology, Fifth Edition.

As used herein, the terms “comprising” (and any form or variant ofcomprising, such as “comprise” and “comprises”), “having” (and any formor variant of having, such as “have” and “has”), “including” (and anyform or variant of including, such as “includes” and “include”), or“containing” (and any form or variant of containing, such as “contains”and “contain”), are inclusive or open-ended and do not excludeadditional, unrecited additives, components, integers, elements ormethod steps.

As used herein, the terms “a,” “an,” and “the” and similar referentsused herein are to be construed to cover both the singular and theplural unless their usage in context indicates otherwise. Accordingly,the use of the word “a” or “an” when used in the claims orspecification, including when used in conjunction with the term“comprising”, may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

As used herein, the terms “link”, “linked”, “linkage” and variantsthereof comprise any type of fusion, bond, adherence or association thatis of sufficient stability to withstand use in the particular biologicalapplication of interest. Such linkage can comprise, for example,covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, oraffinity bonding, bonds or associations involving van der Waals forces,mechanical bonding, and the like. Optionally, such linkage can occurbetween a combination of different molecules, including but not limitedto: between a nanoparticle and a protein; between a protein and a label;between a linker and a functionalized nanoparticle; between a linker anda protein; between a nucleotide and a label; and the like. Some examplesof linkages can be found, for example, in Hermanson, G., BioconjugateTechniques, Second Edition (2008); Aslam, M., Dent, A., Bioconjugation:Protein Coupling Techniques for the Biomedical Sciences, London:Macmillan (1998); Aslam, M., Dent, A., Bioconjugation: Protein CouplingTechniques for the Biomedical Sciences, London: Macmillan (1998).

As used herein, the term “linker” and its variants comprises anycomposition, including any molecular complex or molecular assembly thatserves to link two or more compounds or molecules.

As used herein, the term “polymerase” and its variants comprise anyenzyme that can catalyze the polymerization of nucleotides (includinganalogs thereof) into a nucleic acid strand. Typically but notnecessarily such nucleotide polymerization can occur in atemplate-dependent fashion. Such polymerases can include withoutlimitation naturally-occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids, or the linkage of parts of two ormore polymerases. Typically, the polymerase comprises one or more activesites at which nucleotide binding and/or catalysis of nucleotidepolymerization can occur. Some exemplary polymerases include withoutlimitation DNA polymerases (such as for example Phi-29 DNA polymerase,reverse transcriptases and E. coli DNA polymerase) and RNA polymerases.The term “polymerase” and its variants, as used herein, also refers tofusion proteins comprising at least two portions linked to each other,where the first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide, such as, forexample, a reporter enzyme or a processivity-enhancing domain. Oneexemplary embodiment of such a polymerase is Phusion® DNA polymerase(New England Biolabs), which comprises a Pyrococcus-like polymerasefused to a processivity-enhancing domain as described, for example, inU.S. Pat. No. 6,627,424.

As used herein, the term “polymerase activity” and its variants, whenused in reference to a given polymerase, comprises any in vivo or invitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing the polymerization of nucleotides into a nucleicacid strand, e.g., primer extension activity, and the like. Typically,but not necessarily such nucleotide polymerization occurs in atemplate-dependent fashion. In addition to such polymerase activity, thepolymerase can typically possess other enzymatic activities, forexample, 3′ to 5′ or 5′ to 3′ exonuclease activity.

As used herein, the term “nucleotide” and its variants comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase; occasionallyhowever the nucleotide may dissociate from the polymerase withoutbecoming incorporated into the nucleic acid strand, an event referred toherein as a “non-productive” event. Such nucleotides include not onlynaturally-occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally-occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties. In some embodiments, the nucleotide can optionally include achain of phosphorus atoms comprising three, four, five, six, seven,eight, nine, ten or more phosphorus atoms. In some embodiments, thephosphorus chain can be attached to any carbon of a sugar ring, such asthe 5′ carbon. The phosphorus chain can be linked to the sugar with anintervening O or S. In one embodiment, one or more phosphorus atoms inthe chain can be part of a phosphate group having P and O. In anotherembodiment, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where Rcan be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorusatoms in the chain can have side groups having O, BH3, or S. In thephosphorus chain, a phosphorus atom with a side group other than O canbe a substituted phosphate group. Some examples of nucleotide analogsare described in Xu, U.S. Pat. No. 7,405,281. In some embodiments, thenucleotide comprises a label (e.g., reporter moiety) and referred toherein as a “labeled nucleotide”; the label of the labeled nucleotide isreferred to herein as a “nucleotide label”. In some embodiments, thelabel can be in the form of a fluorescent dye attached to the terminalphosphate group, i.e., the phosphate group or substitute phosphate groupmost distal from the sugar. Some examples of nucleotides that can beused in the disclosed methods and compositions include, but are notlimited to, ribonucleotides, deoxyribonucleotides, modifiedribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, metallonucleosides, phosphonatenucleosides, and modified phosphate-sugar backbone nucleotides, analogs,derivatives, or variants of the foregoing compounds, and the like. Insome embodiments, the nucleotide can comprise non-oxygen moieties suchas, for example, thio- or borano-moieties, in place of the oxygen moietybridging the alpha phosphate and the sugar of the nucleotide, or thealpha and beta phosphates of the nucleotide, or the beta and gammaphosphates of the nucleotide, or between any other two phosphates of thenucleotide, or any combination thereof.

As used herein, the term “nucleotide incorporation” and its variantscomprises polymerization of one or more nucleotides into a nucleic acidstrand.

As used herein, the term “biomolecule” and its variants comprises anycompound isolated from a living organism, as well as analogs (includingengineered and/or synthetic analogs), derivatives, mutants or variantsand/or biologically active fragments of the same. For example, thebiomolecule can be a protein (e.g., enzyme), nucleic acid, nucleotide,carbohydrate or lipid. In some embodiments, the biomolecule can be anengineered or synthetic analog of a compound isolated from a living cellthat is structurally different from the compound but retains abiological activity characteristic of that compound.

As used herein, the term “target” and its variants comprises anycompound that is capable of binding specifically to a particularbiomolecule. In one exemplary embodiment, the target of an enzyme canbe, for example, a substrate of the enzyme.

As used herein, the term “biological activity” and its variants, whenused in reference to a biomolecule (such as, for example, an enzyme)refers to any in vivo or in vitro activity that is characteristic of thebiomolecule itself, including the interaction of the biomolecule withone or more targets. For example, biological activity can optionallyinclude the selective binding of an antibody to an antigen, theenzymatic activity of an enzyme, and the like. Such activity can alsoinclude, without limitation, binding, fusion, bond formation,association, approach, catalysis or chemical reaction, optionally withanother biomolecule or with a target molecule.

As used herein, the term “biologically active fragment” and its variantsrefers to any fragment, derivative or analog of a biomolecule thatpossesses an in vivo or in vitro activity that is characteristic of thebiomolecule itself. For example, the biomolecule can be an antibody thatis characterized by antigen-binding activity, or an enzyme characterizedby the ability to catalyze a particular biochemical reaction, etc.Biologically active fragments can optionally exist in vivo, such as, forexample, fragments which arise from post transcriptional processing orwhich arise from translation of alternatively spliced RNAs, oralternatively can be created through engineering, bulk synthesis, orother suitable manipulation. Biologically active fragments includefragments expressed in native or endogenous cells as well as those madein expression systems such as, for example, in bacterial, yeast, insector mammalian cells. Because biomolecules often exhibit a range ofphysiological properties and because such properties can be attributableto different portions of the biomolecule, a useful biologically activefragment can be a fragment of a biomolecule that exhibits a biologicalactivity in any biological assay. In some embodiments, the fragment oranalog possesses 10%, 40%, 60%, 70%, 80% or 90% or greater of theactivity of the biomolecule in any in vivo or in vitro assay ofinterest.

The term “modification” or “modified” and their variants, as used hereinwith reference to a protein, comprise any change in the structural,biological and/or chemical properties of the protein, particularly achange in the amino acid sequence of the protein. In some embodiments,the modification can comprise one or more amino acid mutations,including without limitation amino acid additions, deletions andsubstitutions (including both conservative and non-conservativesubstitutions).

The terms “resonance energy transfer” and “RET” and their variants, asused herein, refer to a radiationless transmission of excitation energyfrom a first moiety, termed a donor moiety, to a second moiety termed anacceptor moiety. One type of RET includes Forster Resonance EnergyTransfer (FRET), in which a fluorophore (the donor) in an excited statetransfers its energy to a proximal molecule (the acceptor) bynonradiative dipole-dipole interaction. See, e.g., Forster, T.“Intermolecular Energy Migration and Fluorescence”, Ann. Phys 2:55-75,1948; Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 2nd ed.Plenum, New York. 367-394., 1999. RET also comprises luminescenceresonance energy transfer, bioluminescence resonance energy transfer,chemiluminescence resonance energy transfer, and similar types of energytransfer not strictly following the Forster's theory, such asnonoverlapping energy transfer occurring when nonoverlapping acceptorsare utilized. See, for example, Anal. Chem. 2005, 77: 1483-1487.

The term “conservative” and its variants, as used herein with referenceto any change in amino acid sequence, refers to an amino acid mutationwherein one or more amino acids is substituted by another amino acidhaving highly similar properties. For example, one or more amino acidscomprising nonpolar or aliphatic side chains (for example, glycine,alanine, valine, leucine, isoleucine or proline) can be substituted foreach other. Similarly, one or more amino acids comprising polar,uncharged side chains (for example, serine, threonine, cysteine,methionine, asparagine or glutamine) can be substituted for each other.Similarly, one or more amino acids comprising aromatic side chains (forexample, phenylalanine, tyrosine or tryptophan) can be substituted foreach other. Similarly, one or more amino acids comprising positivelycharged side chains (for example, lysine, arginine or histidine) can besubstituted for each other. Similarly, one or more amino acidscomprising negatively charged side chains (for example, aspartic acid orglutamic acid) can be substituted for each other. In some embodiments,the modified polymerase is a variant that comprises one or more of theseconservative amino acid substitutions, or any combination thereof. Insome embodiments, conservative substitutions for leucine include:alanine, isoleucine, valine, phenylalanine, tryptophan, methionine, andcysteine. In other embodiments, conservative substitutions forasparagine include: arginine, lysine, aspartate, glutamate, andglutamine.

The term “primer extension activity” and its variants, as used herein,when used in reference to a given polymerase, comprises any in vivo orin vitro enzymatic activity characteristic of a given polymerase thatrelates to catalyzing nucleotide incorporation onto the terminal 3′OHend of an extending nucleic acid molecule. Typically but not necessarilysuch nucleotide incorporation occurs in a template-dependent fashion.The primer extension activity is typically quantified as the totalnumber of nucleotides incorporated (as measured by, e.g., radiometric orother suitable assay) by a unit amount of polymerase (in moles) per unittime (seconds) under a particular set of reaction conditions.

The terms “His tag” or “His-tag” and their variants as used hereinrefers to a stretch of amino acids comprising multiple histidineresidues. Typically, the His tag can bind to metal ions, for example,Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺ ions. Optionally, the His tag comprises 2, 3,4, 5, 6, 7, 8 or more histidine residues. In some embodiments, the Histag is fused to the N- or C-terminus of a protein; alternatively, it canbe fused at any suitable location within the protein.

As used herein, the term “binding pair” or “binding partner” and itsvariants refers to two molecules, or portions thereof, which have aspecific binding affinity for one another and typically will bind toeach other in preference to binding to other molecules. Typically butnot necessarily some or all of the structure of one member of a specificbinding pair is complementary to some or all of the structure possessedby the other member, with the two members being able to bind togetherspecifically by way of a bond between the complementary structures,optionally by virtue of multiple noncovalent attractions. The twomembers of a binding pair are referred to herein as the “first member”and the “second member” respectively. The following may be mentioned asnon-limiting examples of molecules that can function as a member of aspecific binding pair, without this being understood as any restriction:thyroxin-binding globulin, steroid-binding proteins, antibodies,antigens, haptens, enzymes, lectins, nucleic acids, repressors,oligonucleotides, polynucleotides, protein A, protein G, avidin,streptavidin, biotin, complement component Clq, nucleic acid-bindingproteins, receptors, carbohydrates, complementary nucleic acidsequences, and the like. Examples of specific binding pairs includewithout limitation: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunogically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A;and an oligonucleotide or polynucleotide and its correspondingcomplement.

As used herein, the term “biotin” and its variants comprises biotin(cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) and anyderivatives and analogs thereof, including biotin-like compounds. Suchcompounds include, for example, biotin-e-N-lysine, biocytin hydrazide,amino or sulfhydryl derivatives of 2-iminobiotin andbiotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester,sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide, p-diazobenzoylbiocytin, 3-(N-maleimidopropionyl)biocytin, and the like. “Biotinmoiety” also comprises biotin variants that can specifically bind to anavidin moiety.

The term “biotinylated” and its variants, as used herein, refer to anycovalent or non-covalent adduct of biotin with other moieties such asbiomolecules, e.g., proteins, nucleic acids (including DNA, RNA, DNA/RNAchimeric molecules, nucleic acid analogs and peptide nucleic acids),proteins (including enzymes, peptides and antibodies), carbohydrates,lipids, etc.

The terms “avidin” and “avidin moiety” and their variants, as usedherein, comprises the native egg-white glycoprotein avidin, as well asany derivatives, analogs and other non-native forms of avidin, that canspecifically bind to biotin moieties. In some embodiments, the avidinmoiety can comprise deglycosylated forms of avidin, bacterialstreptavidins produced by selected strains of Streptomyces, e.g.,Streptomyces avidinii, to truncated streptavidins, and to recombinantavidin and streptavidin as well as to derivatives of native,deglycosylated and recombinant avidin and of native, recombinant andtruncated streptavidin, for example, N-acyl avidins, e.g., N-acetyl,N-phthalyl and N-succinyl avidin, and the commercial productsExtrAvidin®, Captavidin®, Neutravidin® and Neutralite Avidin®. All formsof avidin-type molecules, including both native and recombinant avidinand streptavidin as well as derivatized molecules, e.g. nonglycosylatedavidins, N-acyl avidins and truncated streptavidins, are encompassedwithin the terms “avidin” and “avidin moiety”. Typically, but notnecessarily, avidin exists as a tetrameric protein, wherein each of thefour tetramers is capable of binding at least one biotin moiety.

As used herein, the term “biotin-avidin bond” and its variants refers toa specific linkage formed between a biotin moiety and an avidin moiety.Typically, a biotin moiety can bind with high affinity to an avidinmoiety, with a dissociation constant K_(d) typically in the order of10⁻¹⁴ to 10⁻¹⁵ mol/L. Typically, such binding occurs via non-covalentinteractions.

As used herein, the term “accessory compound” and its variants refer toany non-polymerase compound capable of attaching to a nanoparticlethrough one or more attachment sites. Optionally, the accessory compoundcan comprise a His tag.

As used herein, the term “modification enzyme recognition site” refersto an amino acid recognition sequence that is chemically modified in anenzyme-catalyzed reaction, wherein the enzyme catalyzing the reactionexhibits specificity for the amino acid recognition sequence. The aminoacid recognition sequence may be inserted into a protein of interest,for example by conventional recombinant DNA techniques. Examples ofmodification enzyme recognition sites include, but are not limited to abiotin ligase modification site, for example a site comprising the aminoacid sequence GLNDIFEAQKIEWHE (SEQ ID NO: 61), for introducing a biotinmoiety; a protein kinase modification site, for example a sitecomprising the amino acid sequence LRRASLG (SEQ ID NO: 19), forintroducing a phosphorothioate moiety; and a transglutaminasemodification site, for example a site comprising the amino acid sequencePKPQQF (SEQ ID NO: 22), for introducing an amine moiety.

The terms “reporter” and “reporter moiety” and their variants, as usedherein, refer to any moiety that generates, or causes to be generated, adetectable signal. Any suitable reporter moiety may be used, includingluminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent, chromophore,radioisotope, electrochemical, mass spectrometry, Raman, hapten,affinity tag, atom, or an enzyme. The reporter moiety generates, orcauses to be generated, a detectable signal resulting from a chemical orphysical change (e.g., heat, light, electrical, pH, salt concentration,enzymatic activity, or proximity events). A proximity event includes tworeporter moieties approaching each other, or associating with eachother, or binding each other. The appropriate procedures for detecting asignal, or change in the signal, generated by the reporter moiety arewell known in the art. The reporter moieties can be linked to a solidsurface, polymerase, nucleotide (or analog thereof), target nucleic acidmolecule, or primer. In one embodiment, a nucleotide can be linked to areporter moiety. The reporter moiety can generate a signal, or a changein a signal, upon excitation from an appropriated energy source (e.g.,electromagnetic source). Some energy transfer reporter moieties can beoptically or spectrally detectable.

The term “label” and its variants, as used herein, comprises anyoptically detectable moiety and includes any moiety that can be detectedusing, for example, fluorescence, luminescence and/or phosphoresecencespectroscopy, Raman scattering, or diffraction. Exemplary labelsaccording to the present disclosure include fluorescent and luminescentmoieties as well as quenchers thereof. Some typical labels includewithout limitation energy transfer moieties, nanoparticles and organicdyes.

Other objects, features and advantages of the disclosed compositions,methods, systems and kits will become apparent from the followingdetailed description. It should be understood, however, that thedetailed description and the specific examples, while indicatingspecific embodiments, are given by way of illustration only, sincevarious changes and modifications within the spirit and scope of theinventions provided herein will become apparent to those skilled in theart from this detailed description.

Provided herein are methods, compositions, systems and kits fornucleotide polymerization using energy transfer signals, or changes inenergy transfer signals, from an energy transfer system which permitsgenerating, detecting, measuring, and characterizing the energy transfersignals, which are associated with nucleotide polymerization, andpermits identification of nucleotide binding and nucleotideincorporation events. The compositions and methods can permit accuratebase identification for single molecule sequencing reactions.

The methods, compositions, systems and kits provided herein can be usedfor sequence-by-synthesis procedures for deducing the sequence of atarget nucleic acid molecule. The methods permit detection andmonitoring of nucleotide binding and nucleotide polymerization events.The systems comprise polymerases attached with an energy transfer donormoiety (or acceptor moiety), and nucleotides attached with at least oneenergy transfer acceptor moiety (or donor moiety). The donor andacceptor moieties undergo energy transfer when the polymerase andnucleotide are proximal to each other during nucleotide binding and/ornucleotide polymerization. As the donor and acceptor moieties undergoenergy transfer, they generate an energy transfer signal (or a change inan energy transfer signal) which may correlate with nucleotide bindingor polymerization. Detecting a time sequence of the generated energytransfer signals, or the change in the generated energy transfersignals, can be used to determine the order of the incorporatednucleotides, and can therefore be used to deduce the sequence of thetarget molecule.

Nucleotide Polymerization

Provided herein are methods, compositions, systems and kits for:nucleotide binding; nucleotide incorporation; nucleotide polymerization;generating an energy transfer signal which is associated with theproximity of the polymerase and nucleotide; generating an energytransfer signal which is associated with nucleotide incorporation;detecting an energy transfer signal which is associated with nucleotideincorporation; and identifying the energy transfer signal which isassociated with nucleotide incorporation.

The methods include polymerase-dependent nucleotide polymerization in acontinuous (i.e., asynchronous) manner. The compositions, systems,methods and kits comprise polymerases attached with at least one energytransfer donor moiety, and nucleotides attached with at least one energytransfer acceptor moiety. The donor and acceptor moieties undergo energytransfer when the polymerase and nucleotide are proximal to each otherduring nucleotide binding and/or nucleotide incorporation. As the donorand acceptor moieties undergo energy transfer, they generate an energytransfer signal (or a change in an energy transfer signal) which maycorrelate with nucleotide binding to the polymerase or with nucleotideincorporation during polymerization. Detecting the energy transfersignal, or change in the energy transfer signal, can be used to identifythe incorporating nucleotide. Detecting a time sequence of the generatedenergy transfer signals, or the change in the generated energy transfersignals, from successive nucleotide incorporation events can be used todetermine the order of the incorporated nucleotides, and can thereforebe used to deduce the sequence of the target molecule. Nucleotideincorporation includes DNA polymerization and RNA polymerization.

By way of a non-limiting example of nucleotide polymerization, the stepsor events of DNA polymerization are well known and comprise: (1)complementary base-pairing a target DNA molecule (e.g., a templatemolecule) with a DNA primer molecule having a terminal 3′ OH (theterminal 3′ OH provides the polymerization initiation site for DNApolymerase); (2) binding the base-paired target DNA/primer duplex with aDNA-dependent polymerase to form a complex (e.g., open complex); (3) acandidate nucleotide binds with the DNA polymerase which interrogatesthe candidate nucleotide for complementarity with the templatenucleotide on the target DNA molecule; (4) the DNA polymerase mayundergo a conformational change (e.g., to a closed complex if thecandidate nucleotide is complementary); (5) the polymerase catalyzesnucleotide polymerization.

In one embodiment, the polymerase catalyzes nucleotide incorporation.For example, the polymerase catalyzes bond formation between thecandidate nucleotide and the nucleotide at the terminal end of thepolymerization initiation site. The polymerase can catalyze the terminal3′ OH of the primer exerting a nucleophilic attack on the bond betweenthe α and β phosphates of the candidate nucleotide to mediate anucleotidyl transferase reaction resulting in phosphodiester bondformation between the terminal 3′ end of the primer and the candidatenucleotide (i.e., nucleotide incorporation in a template-dependentmanner), and concomitant cleavage to form a cleavage product. Thepolymerase can liberate the cleavage product. In some embodiments, wherethe polymerase incorporates a nucleotide having phosphate groups, thecleavage product includes one or more phosphate groups. In otherembodiments, where the polymerase incorporates a nucleotide havingsubstituted phosphate groups, the cleavage product may include one ormore substituted phosphate groups.

The candidate nucleotide may or may not be complementary to the templatenucleotide on the target molecule. The candidate nucleotide candissociate from the polymerase. If the candidate nucleotide dissociatesfrom the polymerase, it can be liberated and typically carries intactpolyphosphate groups. When the candidate nucleotide dissociates from theDNA polymerase, the event is known as a “non-productive binding” event.The dissociating nucleotide may or may not be complementary to thetemplate nucleotide on the target molecule.

The incorporated nucleotide may or may not be complementary to thetemplate nucleotide on the target. When the candidate nucleotide bindsthe DNA polymerase and is incorporated, the event is a “productivebinding” event. The incorporated nucleotide may or may not becomplementary to the template nucleotide on the target molecule.

The length of time, frequency, or duration of the binding of thecomplementary candidate nucleotide to the polymerase can differ fromthat of the non-complementary candidate nucleotide. This time differencecan be used to distinguish between the complementary andnon-complementary nucleotides, and/or can be used to identify theincorporated nucleotide, and/or can be used to deduce the sequence ofthe target molecule.

The energy transfer signal (or change in energy transfer signal)generated by the energy transfer donor and/or acceptor can be detectedbefore, during, and/or after any nucleotide incorporation event.

Nucleotide incorporation also includes RNA polymerization which may notrequire a primer to initiate nucleotide polymerization. Nucleotideincorporation events involving RNA polymerization are well known in theart.

Productive and Non-Productive Binding

The methods, compositions, systems and kits disclosed herein can be usedfor distinguishing between the productive and non-productive bindingevents. The compositions and methods can also provide base identityinformation during nucleotide incorporation. The compositions includenucleotides and polymerases each attached to at least one energytransfer moiety.

In a productive binding event, the nucleotide can bind/associate withthe polymerase for a time period which is distinguishable (e.g., longeror shorter time period), compared to a non-productive binding event. Ina non-productive binding event, the nucleotide can bind/associate withthe polymerase and then dissociate. The donor and acceptor energytransfer moieties produce detectable energy transfer signals when theyare in proximity to each other and can be associated with productive andnon-productive binding events. Thus, the time-length difference betweensignals from the productive and non-productive binding events canprovide distinction between the two types of events. Typically, thelength of time for a productive binding event is longer compared thelength of time for a non-productive event.

The detectable signals can be classified into true positive and falsepositive signals. For example, the true positive signals can arise fromproductive binding in which the nucleotide binds the polymerase and isincorporated. The incorporated nucleotide can be complementary to thetemplate nucleotide. In another example, the false positive signals canarise from different binding events, including: non-specific binding,non-productive binding, and any event which brings the energy transferdonor and acceptor into sufficient proximity to induce a detectablesignal, but the nucleotide is not incorporated.

Nucleotide Polymerization Reactions and Methods

The methods, compositions, systems and kits disclosed herein can be usedfor single molecule nucleic acid sequencing, by generating an energytransfer signal which is associated with nucleotide incorporation,detecting the generated energy transfer signal, measuring the generatedenergy transfer signal, characterizing the generated energy transfersignal, and identifying the incorporated nucleotide based on thecharacterized energy transfer signal.

Certain embodiments of the methods, composition, systems, and kits offerone or more advantages over other single molecule sequencing methods(see e.g., Korlach U.S. Pat. Nos. 7,033,764; 7,052,847; 7,056,661;7,056,676; 7,361,466; and Hardin U.S. Pat. No. 7,329,492), although noindividual embodiment necessarily displays all advantages. Theadvantages of the energy transfer system and methods include: (1) energytransfer methods, which require very small distances (about 5-10 nm)between the polymerase and nucleotide, to generate the energy transfersignals which are associated with the close proximity of the polymeraseand nucleotide or are associated with nucleotide polymerization, ratherthan signals which are associated with non-productive binding events;(2) conjugates having a polymerase linked to an energy transfer moiety(e.g., donor moiety) in which the polymerase is enzymatically active;(3) polymerases having altered kinetics for nucleotide binding and/ornucleotide incorporation (e.g., U.S. Ser. Nos. 61/242,771 and61/293,618) to improve distinction between productive and non-productivenucleotide binding events compared to polymerases traditionally used fornucleotide polymerization reactions; (4) polymerases having alteredkinetics for nucleotide binding and/or nucleotide incorporation (e.g.,U.S. Ser. Nos. 61/242,771 and 61/293,618) used in combination withlabeled nucleotides having six or more phosphate groups (or substitutedphosphate groups), which improve distinction between productive andnon-productive binding events compared to polymerases and triphosphatenucleotides, which are traditionally used for nucleotide polymerizationreactions; and (5) polymerases having improved photo-stability whenexposed to electromagnetic energy (e.g., exposed to light during thenucleotide incorporation reactions) compared to polymerasestraditionally used for nucleotide polymerization reactions.

The methods can be practiced using suitable conditions which mediatebinding a nucleotide to the polymerase and/or which mediate nucleotideincorporation. The suitable conditions can include: any conjugate havinga polymerase linked to at least one energy transfer moiety (e.g., donormoiety) in which the polymerase is enzymatically active; polymerasesand/or nucleotides which improve distinction between productive andnon-productive nucleotide binding events; and/or polymerases havingimproved photo-stability when exposed to electromagnetic energy (e.g.,light).

The methods provided herein are performed under any conditions which aresuitable for: forming the complex (target/polymerase ortarget/initiation site/polymerase); binding the nucleotide to thepolymerase; permitting the energy transfer and reporter moieties togenerate detectable energy transfer signals when the nucleotide bindsthe polymerase; incorporating the nucleotide; permitting the energytransfer and reporter moieties to generate an energy transfer signalupon close proximity and/or upon nucleotide binding or nucleotideincorporation; detecting the energy transfer signal, or change in theenergy transfer signal, from the energy transfer or reporter moieties;measuring the energy transfer signal; and/or translocation of thepolymerase to the next position on the target molecule.

The suitable conditions include parameters for time, temperature, pH,reagents, buffers, reagents, salts, co-factors, nucleotides, target DNA,primer DNA, enzymes such as nucleic acid-dependent polymerase, amountsand/or ratios of the components in the reactions, and the like. Thereagents or buffers can include a source of monovalent cations, such asKCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate.The reagents or buffers can include a source of divalent cations, suchas Mg²⁻ and/or Mn²⁺, MgCl₂, or Mg-acetate. The buffer can include Tris,Tricine, HEPES, MOPS, ACES, or MES, which can provide a pH range ofabout 5.0 to about 9.5. The buffer can include chelating agents such asEDTA and EGTA, and the like. The suitable conditions can also includecompounds which reduce photo-damage.

Divalent Cations

The methods, compositions, systems and kits disclosed herein can includeany combination of divalent cations. The divalent cations can includeany cation which permits nucleotide binding and/or nucleotideincorporation, including for example: manganese, magnesium, cobalt,strontium, or barium. The divalent cations can include any cation whichpromotes the formation and/or stability of the closed complex(polymerase/target/nucleotide), including magnesium, manganese, andchromium. The divalent cations can include any cation which permitsnucleotide binding to the polymerase but inhibits nucleotideincorporation (e.g., calcium). The divalent cations can include chlorideor acetate forms, including MnCl₂, Mn-acetate, MgCl₂, Mg-acetate, andthe like.

In practicing the nucleotide incorporation methods, some polymerasesexhibit improved nucleotide binding and/or nucleotide incorporationkinetics when used with (i) manganese and/or magnesium, and/or with (ii)tri-, tetra-, penta-, hexa-, or hepta-phosphate nucleotides. In oneembodiment, the disclosed nucleotide incorporation methods can bepracticed using manganese or magnesium, or a combination of manganeseand magnesium. For example, the methods can include manganese at about0.1-5 mM, or about 0.2-4 mM, or about 0.3-3 mM, or about 0.4-2 mM, orabout 0.5-2 mM, or about 1-2 mM.

In another example, the methods can include magnesium at about 0.01-0.3mM, or about 0.025-0.2 mM, or about 0.05-0.1 mM, or about 0.075-0.1 mM,or about 0.1 mM.

In yet another example, the methods can include a combination ofmanganese and magnesium at about 0.25-1 mM of manganese and 0.025-0.2 mMof magnesium, or about 0.5-0.75 mM of manganese and 0.05-0.075 mM ofmagnesium, or about 0.5 mM manganese and 0.1 mM magnesium.

In another example, the nucleotide incorporation reaction include B103polymerase (SEQ ID NOS:1, 2 or 3) and labeled hexa-phosphatenucleotides, with about 0.5-2 mM MnCl₂, or with a combination of about0.5 mM MnCl₂ and about 0.1 mM MgCl₂.

Polymerization Initiation Sites

The methods, compositions, systems and kits disclosed herein can includea polymerization initiation site. The polymerization initiation site canbe used by the polymerase (e.g., DNA or RNA polymerase) to initiatenucleotide polymerization. In some embodiments, the polymerizationinitiation site can be a terminal 3′ OH group. The 3′ OH group can serveas a substrate for the polymerase for nucleotide polymerization. The 3′OH group can serve as a substrate for the polymerase to form aphosphodiester bond between the terminal 3′ OH group and an incorporatednucleotide. The 3′ OH group can be provided by: the terminal end of aprimer molecule; a nick or gap within a nucleic acid molecule (e.g.,oligonucleotide) which is base-paired with the target molecule; theterminal end of a secondary structure (e.g., the end of a hairpin-likestructure); or an origin of replication. The polymerization initiationsite can be provided by an accessory protein (e.g., RNA polymerase orhelicase/primase). The polymerization initiation site can be provided bya terminal protein which can be bound (covalently or non-covalently) tothe end of the target nucleic acid, including terminal protein (e.g.,TP) found in phage (e.g., TP from phi29 phage). Thus, the polymerizationinitiation site may be at a terminal end or within a base-paired nucleicacid molecule. In other embodiments, the polymerization initiation siteused by some polymerases (e.g., RNA polymerase) may not include a 3′OHgroup.

The portion of the target molecule which is base paired with the primeror with the oligonucleotide, or the self-primed portion of the targetmolecule, can form hydrogen bonding by Watson-Crick or Hoogstein bindingto form a duplex nucleic acid structure. The primer, oligonucleotide,and self-priming sequence may be complementary, or partiallycomplementary, to the nucleotide sequence of the target molecule. Thecomplementary base pairing can be the standard A-T or C-G base pairing,or can be other forms of base-pairing interactions.

The polymerization initiation site can be in a position on the targetnucleic acid molecule to permit nucleotide incorporation events in adirection away from, or towards, the solid surface.

Some polymerases exhibit a preference for binding single-strandednucleic acid molecules. For example, multiple polymerases maypreferentially bind the single-stranded portion of a target nucleic acidmolecule which is base-paired with a primer. In cases where one targetmolecule is bound by multiple polymerases, the efficiency ofpolymerization initiation can be poor. Initiating nucleotidepolymerization using a gap can improve the number of target nucleic acidmolecules which can undergo polymerization. In one embodiment, anunexpected procedure for improving a nucleotide polymerization caninclude initiating the polymerization reaction with the terminal 3′OHwithin a gap, rather than from a primer (which is base-paired with thetarget molecule). In one embodiment, the polymerases which can initiatenucleotide polymerization from a gap include strand-displacingpolymerases. For example, the strand-displacing polymerase can be aphi29-like polymerases including: phi29, B103 (SEQ ID NO: 1, 2 or 3),and GA-1. In one embodiment, the gap can be the length of apolynucleotide molecule which is about 2-15 nucleotides in length, orabout 3-14 in length, or about 4-13 in length, or about 5-12 in length,or about 6-11 in length, or about 7-10 in length. The gap can be formedby annealing a target nucleic acid molecule to two primer nucleic acidmolecules. Forming a gap is well known in the art.

Primer Molecules

The methods, compositions, systems and kits disclosed herein can includea primer molecule which can hybridize with the target nucleic acidmolecule. The sequence of the primer molecule can be complementary ornon-complementary with the sequence of the sequence of the targetmolecule. The terminal 3′ OH of the primer molecule can provide thepolymerization initiation site.

The primers can be modified with a chemical moiety to protect the primerfrom serving as a polymerization initiation site or as a restrictionenzyme recognition site. The chemical moiety can be a natural orsynthetic amino acid linked through an amide bond to the primer.

The primer, oligonucleotide, or self-priming portion, may benaturally-occurring, or may be produced using enzymatic or chemicalsynthesis methods. The primer, oligonucleotide, or self-priming portionmay be any suitable length including 5, 10, 15, 20, 25, 30, 40, 50, 75,or 100 nucleotides or longer in length. The primer, oligonucleotide, orself-priming portion may be linked to an energy transfer moiety (e.g.,donor or acceptor) or to a reporter moiety (e.g., a dye) using methodswell known in the art.

The primer molecule, oligonucleotide, and self-priming portion of thetarget molecule, may comprise ribonucleotides, deoxyribonucleotides,ribonucleotides, deoxyribonucleotides, peptide nucleotides, modifiedphosphate-sugar backbone nucleotides including phosphorothioate andphosphoramidate, metallonucleosides, phosphonate nucleosides, and anyvariants thereof, or combinations thereof.

In one embodiment, the primer molecule can be a recombinant DNAmolecule. The primer can be linked at the 5′ or 3′ end, or internally,with at least one binding partner, such as biotin. The biotin can beused to immobilize the primer molecule to the surface (via anavidin-like molecule), or for attaching the primer to a reporter moiety.The primer can be linked to at least one energy transfer moiety, such asa fluorescent dye or a nanoparticle, or to a reporter moiety. The primermolecule can hybridize to the target nucleic acid molecule. The primermolecule can be used as a capture probe to immobilize the targetmolecule.

Reducing Photo-Damage

The methods, compositions, systems and kits disclosed herein can includecompounds which reduce oxygen-damage or photo-damage. Illuminating thenucleotide binding and/or nucleotide incorporation reactions withelectromagnetic radiation can induce formation of reactive oxygenspecies from the fluorophore or other components in the reaction. Thereactive oxygen species can cause photo-damage to the fluorophores,polymerases, or any other component of the binding or incorporationreactions. The nucleotide binding or nucleotide incorporation reactionscan include compounds which are capable of reducing photo-damage,including: protocatechuate-3,4-dioxygenase, protocatechuic acid;6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (TROLOX); orcyclooctatetraene (COT).

Other compounds for reducing photo-damage include: ascorbic acid,astazanthin, bilirubin, biliverdin, bixin, captopril, canthazanthin,carotene (alpha, beta, and gamma), cysteine, beta-dimethyl cysteine,N-acetyl cysteine, diazobicyclooctane (DABCO), dithiothreitol (DTT),ergothioneine, glucose oxidase/catalase (GO/Cat), glutathione,glutathione peroxidase, hydrazine (N₂H₄), hydroxylamine, lycopene,lutein, polyene dialdehydes, melatonin, methionine,mercaptopropionylglycine, 2-mercaptoethane sulfonate (MESNA), pyridoxine1 and its derivatives, mercaptoethylamine (MEA), β-mercaptoethanol(BME), n-propyl gallate, p-phenylenediamene (PPD), hydroquinone, sodiumazide (NaN₃), sodium sulfite (Na₂SO₃), superoxide dismutase,tocopherols, α-tocopheryl succinate and its analogs, and zeaxanthin.

Methods for Generating an Energy Transfer Signal: Proximity

The methods, compositions, systems and kits disclosed herein can be usedfor generating a signal which is associated with close proximity of thepolymerase and nucleotide.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) at least onetype of a nucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide into the nucleic acid molecule therebylocating the polymerase and nucleotide in close proximity with eachother to generate the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) at least onetype of a hexaphosphate nucleotide having an energy transfer acceptormoiety, so as to incorporate the hexaphosphate nucleotide into thenucleic acid molecule thereby locating the polymerase and nucleotide inclose proximity with each other to generate the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a target nucleic acid molecule which is base-pairedwith a polymerization initiation site having a 3′ OH group and with(iii) at least one type of a nucleotide having an energy transferacceptor moiety, so as to incorporate the nucleotide onto the 3′ OHgroup thereby locating the polymerase and nucleotide in close proximitywith each other to generate the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a target nucleic acid molecule which is base-pairedwith a polymerization initiation site having a 3′ OH group and with(iii) at least one type of a hexaphosphate nucleotide having an energytransfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide onto the 3′ OH group thereby locating the polymerase andnucleotide in close proximity with each other to generate the energytransfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a nucleic acid molecule and with(iii) at least one type of a nucleotide having an energy transferacceptor moiety, so as to incorporate the nucleotide into the nucleicacid molecule thereby locating the polymerase and nucleotide in closeproximity with each other to generate the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a nucleic acid molecule and with(iii) at least one type of a hexaphosphate nucleotide having an energytransfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide into the nucleic acid molecule thereby locating thepolymerase and nucleotide in close proximity with each other to generatethe energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a target nucleic acid moleculewhich is base-paired with a polymerization initiation site having a 3′OH group and with (iii) at least one type of a nucleotide having anenergy transfer acceptor moiety, so as to incorporate the nucleotideonto the 3′ OH group thereby locating the polymerase and nucleotide inclose proximity with each other to generate the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a target nucleic acid moleculewhich is base-paired with a polymerization initiation site having a 3′OH group and with (iii) at least one type of a hexaphosphate nucleotidehaving an energy transfer acceptor moiety, so as to incorporate thehexaphosphate nucleotide onto the 3′ OH group thereby locating thepolymerase and nucleotide in close proximity with each other to generatethe energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase according to anyone of SEQ ID NO:1-3 and linked to an energy transfer donor moiety with(ii) a nucleic acid molecule and with (iii) at least one type of ahexaphosphate nucleotide linked to an energy transfer acceptor moiety,so as to incorporate the hexaphosphate nucleotide into the nucleic acidmolecule thereby locating the polymerase and nucleotide in closeproximity with each other to generate the energy transfer signal.

Detecting the Energy Transfer Signal

In one embodiment, additional steps can be conducted to detect theenergy transfer signal or the change in the energy transfer signal. Theadditional steps comprise: (a) exciting the energy transfer donor moietywith an excitation source; and (b) detecting the energy transfer signalor a change in the energy transfer signal from the energy transfer donormoiety and the energy transfer acceptor moiety that are in closeproximity to each other.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be a laser. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal is a FRET signal. In yet another embodiment, the energytransfer signal or the change in the energy transfer signal can beoptically detectable.

Identifying the Incorporated Nucleotide

In another embodiment, additional steps can be conducted to identify theenergy transfer signal, which can also identify the incorporatednucleotide. The additional steps comprise: (a) exciting the energytransfer donor moiety with an excitation source; (b) detecting theenergy transfer signal or a change in the energy transfer signal fromthe energy transfer donor moiety and the energy transfer acceptor moietythat are in close proximity to each other; and (c) identifying theenergy transfer signal or the change in the energy transfer signal fromthe energy transfer accepter moiety.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be a laser. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal is a FRET signal. In yet another embodiment, the energytransfer signal or the change in the energy transfer signal can beoptically detectable.

Embodiments of Methods for Generating an Energy Transfer Signal:Proximity

In methods for generating an energy transfer signal, in one embodiment,the energy transfer donor and acceptor moieties can fluoresce inresponse to exposure to an excitation source, such as electromagneticradiation. These fluorescence responses can be an energy transfersignal. In another embodiment, the energy transfer acceptor moiety canfluoresce in response to energy transferred from a proximal excitedenergy transfer donor moiety. These fluorescence responses can be anenergy transfer signal. The proximal distance between the donor andacceptor moieties that accommodates energy transfer can be dependentupon the particular donor/acceptor pair. The proximal distance betweenthe donor and acceptor moieties can be about 1-20 nm, or about 1-10 nm,or about 1-5 nm, or about 5-10 nm. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety can remain unchanged. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety results in changes in the energy transfer signal. Inanother embodiment, the changes in the energy transfer signals from thedonor or acceptor moiety can include changes in the: intensity of thesignal; duration of the signal; wavelength of the signal; amplitude ofthe signal; polarization state of the signal; duration between thesignals; and/or rate of the change in intensity, duration, wavelength oramplitude. In another embodiment, the change in the energy transfersignal can include a change in the ratio of the change of the energytransfer donor signal relative to change of the energy transfer acceptorsignals. In another embodiment, the energy transfer signal from thedonor can increase or decrease. In another embodiment, the energytransfer signal from the acceptor can increase or decrease. In anotherembodiment, the energy transfer signal associated with nucleotideincorporation includes: a decrease in the donor signal when the donor isproximal to the acceptor; an increase in the acceptor signal when theacceptor is proximal to the donor; an increase in the donor signal whenthe distance between the donor and acceptor increases; and/or a decreasein the acceptor signal when the distance between the donor and acceptorincreases.

In one embodiment, the detecting the energy transfer signal can beperformed using confocal laser scanning microscopy, Total InternalReflection (TIR), Total Internal Reflection Fluorescence (TIRF),near-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, and/or multi-foci multi-photon.

In practicing the nucleotide binding and/or nucleotide incorporationmethods, non-desirable fluorescent signals can come from sourcesincluding background and/or noise. In one embodiment, the energytransfer signals can be distinguished from the non-desirable fluorescentsignals by measuring, analyzing and characterizing attributes of allfluorescent signals generated by the nucleotide incorporation reaction.In one embodiment, attributes of the energy transfer signal that canpermit distinction from the non-desirable fluorescent signals caninclude: duration; wavelength; amplitude; photon count; and/or the rateof change of the duration, wavelength, amplitude; and/or photon count.In one embodiment, the identifying the energy transfer signal, includesmeasuring, analyzing and characterizing attributes of: duration;wavelength; amplitude; photon count and/or the rate of change of theduration, wavelength, amplitude; and/or photon count. In one embodiment,identifying the energy transfer signal can be used to identify theincorporated nucleotide.

In one embodiment, the nucleic acid molecule can be DNA, RNA or DNA/RNA.

In one embodiment, the polymerase has an active site. The nucleotide canbind the active site. In another embodiment, the polymerase can be aDNA-dependent or RNA-dependent polymerase, or a reverse transcriptase.In another embodiment, the polymerase having altered nucleotide bindingand/or nucleotide incorporation kinetics can improve distinction betweenproductive and non-productive binding events. In another embodiment, thealtered nucleotide binding kinetics and/or altered nucleotideincorporation kinetics can include altered kinetics for: polymerasebinding to the target molecule; polymerase binding to the nucleotide;polymerase catalyzing nucleotide incorporation; the polymerase cleavingthe nucleotide and forming a cleavage product; and/or the polymerasereleasing the cleavage product. In another embodiment, the polymerasecan be linked to an energy transfer donor moiety to form a conjugate. Inanother embodiment, the polymerase component of the conjugate can beenzymatically active. In another embodiment, the polymerase has alteredkinetics for nucleotide binding and/or nucleotide incorporation used incombination with labeled nucleotides having six or more phosphate groups(or substituted phosphate groups), which improve distinction betweenproductive and non-productive binding events. In another embodiment, thepolymerase can have improved photo-stability. The polymerase can be aPhi29-like polymerase, including Phi29 or B103 polymerase. Thepolymerase can be a mutant polymerase. The polymerase can be a B103polymerase according to any one of SEQ ID NOS: 1-5.

In one embodiment, the energy transfer donor moiety can be ananoparticle or a fluorescent dye. The nanoparticle can be about 1-20 nmin its largest dimensions. The nanoparticle can be a core/shellnanoparticle. The nanoparticle can include a core comprisingsemiconductor material(s). The core can include materials (includingbinary, ternary and quaternary mixtures thereof), from: Groups II-VI ofthe periodic table, including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, HgTe, MgTe; Groups III-V, including GaN, GaP, GaAs, GaSb, InN,InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/or Group IV, including Ge,Si, Pb. The nanoparticle can include at least one shell surrounding thecore. The shell can include semiconductor material(s). The nanoparticlecan include an inner shell and an outer shell. The shell can includematerials (including binary, ternary and quaternary mixtures thereof)comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs,GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs,AlN, AlP, or AlSb. In one embodiment, the nanoparticle comprises a corehaving CdSe. In another embodiment, the nanoparticle comprises an innershell having CdS. In another embodiment, the nanoparticle comprises anouter shell having ZnS. The outermost surface of the core or shell canbe coated with tightly associated ligands which are not removed byordinary solvation. In some embodiments, the nanoparticle can have alayer of ligands on its surface which can further be cross-linked toeach other. In some embodiments, the nanoparticle can have other oradditional surface coatings which can modify the properties of theparticle, for example, increasing or decreasing solubility in water orother solvents. The nanoparticle can be water dispersible. Thenanoparticle can be a non-blinking nanoparticle. The nanoparticle can bephoto-stable. The nanoparticle may not interfere with polymeraseactivity, including polymerase binding to the target molecule,polymerase binding to the nucleotide, polymerase catalyzing nucleotideincorporation, or the polymerase cleaving the nucleotide and/orreleasing the cleavage product.

In one embodiment, the target nucleic acid molecule can be DNA or RNA orDNA/RNA molecule. In another embodiment, the target nucleic acidmolecule is a single nucleic acid molecule. In another embodiment, thetarget nucleic acid molecule (e.g., target molecule) is base-paired witha polymerization initiation site. In another embodiment, thepolymerization initiation site is a terminal 3′OH of a primer moleculeor of a self-primed target molecule. In another embodiment, thepolymerization initiation site is a 3′OH within a gap or nick. Inanother embodiment, the target nucleic acid molecule and/or thepolymerization initiation site is immobilized to a solid surface. Inanother embodiment, the target nucleic acid molecule is a linear orcircular nucleic acid molecule.

In one embodiment, the at least one type of nucleotide can include 3-10phosphate groups or substituted phosphate groups, or a combination ofphosphate groups and substituted phosphate groups. The nucleotide caninclude a terminal phosphate group or terminal substituted phosphategroup which can be linked to the energy transfer acceptor moiety. Thenucleotide can include the energy transfer acceptor moiety which islinked the base, sugar, or any phosphate group or substituted phosphategroup. The nucleotide can be adenosine, guanosine, cytosine, thymidine,uridine, or any other type of nucleotide.

In one embodiment, the energy transfer acceptor moiety can be afluorescent dye. The energy transfer acceptor moiety and the energytransfer donor moiety can be capable of energy transfer.

In one embodiment, more than one type of nucleotide can be contactedwith the polymerase. Each of the different types of nucleotides can belinked to the same or to different types of energy transfer acceptormoieties, or any combination of the same or different types of acceptormoieties.

Methods for Generating an Energy Transfer Signal: NucleotideIncorporation

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) at least onetype of a nucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide into the nucleic acid molecule therebygenerating the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) at least onetype of a hexaphosphate nucleotide having an energy transfer acceptormoiety, so as to incorporate the hexaphosphate nucleotide into thenucleic acid molecule thereby generating the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a target nucleic acid molecule which is base-pairedwith a polymerization initiation site having a 3′ OH group and with(iii) at least one type of a nucleotide having an energy transferacceptor moiety, so as to incorporate the nucleotide onto the 3′ OHgroup thereby generating the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a target nucleic acid molecule which is base-pairedwith a polymerization initiation site having a 3′ OH group and with(iii) at least one type of a hexaphosphate nucleotide having an energytransfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide onto the 3′ OH group thereby generating the energy transfersignal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a nucleic acid molecule and with(iii) at least one type of a nucleotide having an energy transferacceptor moiety, so as to incorporate the nucleotide into the nucleicacid molecule thereby generating the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a nucleic acid molecule and with(iii) at least one type of a hexaphosphate nucleotide having an energytransfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide into the nucleic acid molecule thereby generating the energytransfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a target nucleic acid moleculewhich is base-paired with a polymerization initiation site having a 3′OH group and with (iii) at least one type of a nucleotide having anenergy transfer acceptor moiety, so as to incorporate the nucleotideonto the 3′ OH group thereby generating the energy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having an energytransfer donor nanoparticle with (ii) a target nucleic acid moleculewhich is base-paired with a polymerization initiation site having a 3′OH group and with (iii) at least one type of a hexaphosphate nucleotidehaving an energy transfer acceptor moiety, so as to incorporate thehexaphosphate nucleotide onto the 3′ OH group thereby generating theenergy transfer signal.

Provided herein are methods for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase according to anyone of SEQ ID NOS:1-3 and linked to an energy transfer donor moiety with(ii) a nucleic acid molecule and with (iii) at least one type of ahexaphosphate nucleotide linked to an energy transfer acceptor moiety,so as to incorporate the hexaphosphate nucleotide into the nucleic acidmolecule thereby generating the energy transfer signal.

Detecting the Energy Transfer Signal

In one embodiment, additional steps can be conducted to detect theenergy transfer signal or the change in the energy transfer signal. Theadditional steps comprise: (a) exciting the energy transfer donor moietywith an excitation source; and (b) detecting the energy transfer signalor a change in the energy transfer signal from the energy transfer donormoiety and the energy transfer acceptor moiety that are in closeproximity to each other.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be a laser. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal is a FRET signal. In yet another embodiment, the energytransfer signal or the change in the energy transfer signal can beoptically detectable.

Identifying the Incorporated Nucleotide

In another embodiment, additional steps can be conducted to identify theincorporated nucleotide. The additional steps comprise: (a) exciting theenergy transfer donor moiety with an excitation source; (b) detectingthe energy transfer signal or a change in the energy transfer signalfrom the energy transfer donor moiety and the energy transfer acceptormoiety that are in close proximity to each other; and (c) identifyingthe energy transfer signal or the change in the energy transfer signalfrom the energy transfer accepter moiety.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be a laser. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal is a FRET signal. In yet another embodiment, the energytransfer signal or the change in the energy transfer signal can beoptically detectable.

Embodiments of Methods for Generating an Energy Transfer Signal:

Nucleotide Incorporation:

In methods for generating an energy transfer signal, in one embodiment,the energy transfer donor and acceptor moieties can fluoresce inresponse to exposure to an excitation source, such as electromagneticradiation. These fluorescence responses can be an energy transfersignal. In another embodiment, the energy transfer acceptor moiety canfluoresce in response to energy transferred from a proximal excitedenergy transfer donor moiety. These fluorescence responses can be anenergy transfer signal. The proximal distance between the donor andacceptor moieties that accommodates energy transfer can be dependentupon the particular donor/acceptor pair. The proximal distance betweenthe donor and acceptor moieties can be about 1-20 nm, or about 1-10 nm,or about 1-5 nm, or about 5-10 nm. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety can remain unchanged. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety results in changes in the energy transfer signal. Inanother embodiment, the changes in the energy transfer signals from thedonor or acceptor moiety can include changes in the: intensity of thesignal; duration of the signal; wavelength of the signal; amplitude ofthe signal; polarization state of the signal; duration between thesignals; and/or rate of the change in intensity, duration, wavelength oramplitude. In another embodiment, the change in the energy transfersignal can include a change in the ratio of the change of the energytransfer donor signal relative to change of the energy transfer acceptorsignals. In another embodiment, the energy transfer signal from thedonor can increase or decrease. In another embodiment, the energytransfer signal from the acceptor can increase or decrease. In anotherembodiment, the energy transfer signal associated with nucleotideincorporation includes: a decrease in the donor signal when the donor isproximal to the acceptor; an increase in the acceptor signal when theacceptor is proximal to the donor; an increase in the donor signal whenthe distance between the donor and acceptor increases; and/or a decreasein the acceptor signal when the distance between the donor and acceptorincreases.

In one embodiment, the detecting the energy transfer signal can beperformed using confocal laser scanning microscopy, Total InternalReflection (TIR), Total Internal Reflection Fluorescence (TIRF),near-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, and/or multi-foci multi-photon.

In practicing the nucleotide binding and/or nucleotide incorporationmethods, non-desirable fluorescent signals can come from sourcesincluding background and/or noise. In one embodiment, the energytransfer signals can be distinguished from the non-desirable fluorescentsignals by measuring, analyzing and characterizing attributes of allfluorescent signals generated by the nucleotide incorporation reaction.In one embodiment, attributes of the energy transfer signal that canpermit distinction from the non-desirable fluorescent signals caninclude: duration; wavelength; amplitude; photon count; and/or the rateof change of the duration, wavelength, amplitude; and/or photon count.In one embodiment, the identifying the energy transfer signal, includesmeasuring, analyzing and characterizing attributes of: duration;wavelength; amplitude; photon count and/or the rate of change of theduration, wavelength, amplitude; and/or photon count. In one embodiment,identifying the energy transfer signal can be used to identify theincorporated nucleotide.

In one embodiment, the nucleic acid molecule can be DNA, RNA or DNA/RNA.

In one embodiment, the polymerase has an active site. The nucleotide canbind the active site. In another embodiment, the polymerase can be aDNA-dependent or RNA-dependent polymerase, or a reverse transcriptase.In another embodiment, the polymerase having altered nucleotide bindingand/or nucleotide incorporation kinetics can improve distinction betweenproductive and non-productive binding events. In another embodiment, thealtered nucleotide binding kinetics and/or altered nucleotideincorporation kinetics can include altered kinetics for: polymerasebinding to the target molecule; polymerase binding to the nucleotide;polymerase catalyzing nucleotide incorporation; the polymerase cleavingthe nucleotide and forming a cleavage product; and/or the polymerasereleasing the cleavage product. In another embodiment, the polymerasecan be linked to an energy transfer donor moiety to form a conjugate. Inanother embodiment, the polymerase component of the conjugate can beenzymatically active. In another embodiment, the polymerase has alteredkinetics for nucleotide binding and/or nucleotide incorporation used incombination with labeled nucleotides having six or more phosphate groups(or substituted phosphate groups), which improve distinction betweenproductive and non-productive binding events. In another embodiment, thepolymerase can have improved photo-stability. The polymerase can be aPhi29-like polymerase, including Phi29 or B103 polymerase. Thepolymerase can be a mutant polymerase. The polymerase can be a B103polymerase according to any one of SEQ ID NOS: 1-5.

In one embodiment, the energy transfer donor moiety can be ananoparticle or a fluorescent dye. The nanoparticle can be about 1-20 nmin its largest dimensions. The nanoparticle can be a core/shellnanoparticle. The nanoparticle can include a core comprisingsemiconductor material(s). The core can include materials (includingbinary, ternary and quaternary mixtures thereof), from: Groups II-VI ofthe periodic table, including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, HgTe, MgTe; Groups III-V, including GaN, GaP, GaAs, GaSb, InN,InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/or Group IV, including Ge,Si, Pb. The nanoparticle can include at least one shell surrounding thecore. The shell can include semiconductor material(s). The nanoparticlecan include an inner shell and an outer shell. The shell can includematerials (including binary, ternary and quaternary mixtures thereof)comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs,GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs,AlN, AlP, or AlSb. In one embodiment, the nanoparticle comprises a corehaving CdSe. In another embodiment, the nanoparticle comprises an innershell having CdS. In another embodiment, the nanoparticle comprises anouter shell having ZnS. The outermost surface of the core or shell canbe coated with tightly associated ligands which are not removed byordinary solvation. In some embodiments, the nanoparticle can have alayer of ligands on its surface which can further be cross-linked toeach other. In some embodiments, the nanoparticle can have other oradditional surface coatings which can modify the properties of theparticle, for example, increasing or decreasing solubility in water orother solvents. The nanoparticle can be water dispersible. Thenanoparticle can be a non-blinking nanoparticle. The nanoparticle can bephoto-stable. The nanoparticle may not interfere with polymeraseactivity, including polymerase binding to the target molecule,polymerase binding to the nucleotide, polymerase catalyzing nucleotideincorporation, or the polymerase cleaving the nucleotide and/orreleasing the cleavage product.

In one embodiment, the target nucleic acid molecule can be DNA or RNA orDNA/RNA molecule. In another embodiment, the target nucleic acidmolecule is a single nucleic acid molecule. In another embodiment, thetarget nucleic acid molecule (e.g., target molecule) is base-paired witha polymerization initiation site. In another embodiment, thepolymerization initiation site is a terminal 3′OH of a primer moleculeor of a self-primed target molecule. In another embodiment, thepolymerization initiation site is a 3′OH within a gap or nick. Inanother embodiment, the target nucleic acid molecule and/or thepolymerization initiation site is immobilized to a solid surface. Inanother embodiment, the target nucleic acid molecule is a linear orcircular nucleic acid molecule.

In one embodiment, the at least one type of nucleotide can include 3-10phosphate groups or substituted phosphate groups, or a combination ofphosphate groups and substituted phosphate groups. The nucleotide caninclude a terminal phosphate group or terminal substituted phosphategroup which can be linked to the energy transfer acceptor moiety. Thenucleotide can include the energy transfer acceptor moiety which islinked the base, sugar, or any phosphate group or substituted phosphategroup. The nucleotide can be adenosine, guanosine, cytosine, thymidine,uridine, or any other type of nucleotide.

In one embodiment, the energy transfer acceptor moiety can be afluorescent dye. The energy transfer acceptor moiety and the energytransfer donor moiety can be capable of energy transfer.

In one embodiment, more than one type of nucleotide can be contactedwith the polymerase. Each of the different types of nucleotides can belinked to the same or to different types of energy transfer acceptormoieties, or any combination of the same or different types of acceptormoieties.

Methods for Incorporating Nucleotides

The methods, compositions, systems and kits disclosed herein can be usedfor incorporating nucleotides. Provided herein are methods forincorporating a nucleotide, comprising: contacting (i) a polymerasehaving altered nucleotide incorporation kinetics and linked to an energytransfer donor moiety with (ii) a nucleic acid molecule and with (iii)at least one type of a nucleotide having an energy transfer acceptormoiety, so as to incorporate the nucleotide into the nucleic acidmolecule. In one embodiment, the nucleic acid molecule includes apolymerization initiation site having a 3′ OH group.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase having altered nucleotideincorporation kinetics and linked to an energy transfer donor moietywith (ii) a nucleic acid molecule and with (iii) at least one type of anucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide into the nucleic acid molecule.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase having altered nucleotideincorporation kinetics and linked to an energy transfer donor moietywith (ii) a nucleic acid molecule and with (iii) at least one type of ahexaphosphate nucleotide having an energy transfer acceptor moiety, soas to incorporate the hexaphosphate nucleotide into the nucleic acidmolecule.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase having altered nucleotideincorporation kinetics and linked to an energy transfer donor moietywith (ii) a target nucleic acid molecule which is base-paired with apolymerization initiation site having a 3′ OH group and with (iii) atleast one type of a nucleotide having an energy transfer acceptormoiety, so as to incorporate the nucleotide onto the 3′OH group.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase having altered nucleotideincorporation kinetics and linked to an energy transfer donor moietywith (ii) a target nucleic acid molecule which is base-paired with apolymerization initiation site having a 3′ OH group and with (iii) atleast one type of a hexaphosphate nucleotide having an energy transferacceptor moiety, so as to incorporate the hexaphosphate nucleotide ontothe 3′OH group.

Provided herein are methods for incorporating a nucleotide, comprisingthe steps of: contacting (i) a polymerase including an energy transferdonor nanoparticle with (ii) a nucleic acid molecule and with (iii) atleast one type of a nucleotide having an energy transfer acceptormoiety, so as to incorporate the nucleotide into the nucleic acidmolecule.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase including an energy transferdonor nanoparticle with (ii) a nucleic acid molecule and with (iii) atleast one type of a hexaphosphate phosphate nucleotide having an energytransfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide into the nucleic acid molecule.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase including an energy transferdonor nanoparticle with (ii) a target nucleic acid molecule which isbase-paired with a polymerization initiation site having a 3′ OH groupand with (iii) at least one type of a nucleotide having an energytransfer acceptor moiety, so as to incorporate the nucleotide onto the3′OH group.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase including an energy transferdonor nanoparticle with (ii) a target nucleic acid molecule which isbase-paired with a polymerization initiation site having a 3′ OH groupand with (iii) at least one type of a hexaphosphate nucleotide having anenergy transfer acceptor moiety, so as to incorporate the hexaphosphatenucleotide onto the 3′OH group.

Provided herein are methods for conducting a plurality of nucleotideincorporation reactions (e.g., arrays), each nucleotide incorporationreaction comprises the steps of: contacting (i) a polymerase havingaltered nucleotide incorporation kinetics and linked to an energytransfer donor moiety with (ii) a nucleic acid molecule and with (iii)at least one type of a nucleotide having an energy transfer acceptormoiety, so as to incorporate the nucleotide into the nucleic acidmolecule.

Provided herein are methods for conducting a plurality of nucleotideincorporation reactions (e.g., arrays), each nucleotide incorporationreaction comprises the steps of: contacting (i) a polymerase includingan energy transfer donor nanoparticle with (ii) a nucleic acid moleculeand with (iii) at least one type of a nucleotide having an energytransfer acceptor moiety, so as to incorporate the nucleotide into thenucleic acid molecule.

Provided herein are methods for successively incorporating nucleotidescomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) a plurality ofmore than one type of a nucleotide each type of nucleotide having adifferent type of energy transfer acceptor moiety, so as to successivelyincorporate the nucleotides into the nucleic acid molecule.

Provided herein are methods for successively incorporating nucleotidescomprising the steps of: contacting (i) a polymerase including an energytransfer donor nanoparticle with (ii) a nucleic acid molecule and with(iii) a plurality of more than one type of a nucleotide each type ofnucleotide having a different type of energy transfer acceptor moiety,so as to successively incorporate the nucleotides into the nucleic acidmolecule.

Provided herein are methods for successively incorporating nucleotides,comprising the steps of: contacting (i) a mutant polymerase havingaltered nucleotide incorporation kinetics and linked to an energytransfer donor moiety with (ii) a target DNA molecule which isbase-paired with a polymerization initiation site having a 3′ OH groupand with (iii) a plurality of more than one type of a hexaphosphatenucleotide each type of hexaphosphate nucleotide having a different typeof fluorescent dye acceptor, so as to successively incorporate thehexaphosphate nucleotides onto the 3′ OH group.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase according to any one of SEQ IDNO:1-3 and linked to an energy transfer donor moiety with (ii) a nucleicacid molecule (iii) at least one type of a hexaphosphate nucleotidelinked to an energy transfer acceptor moiety, so as to incorporate thehexaphosphate nucleotide into the nucleic acid molecule.

Provided herein are methods for incorporating a nucleotide comprisingthe steps of: contacting (i) a polymerase according to any one of SEQ IDNO:1-3 and linked to an energy transfer donor moiety with (ii) a nucleicacid molecule which is base-paired with a polymerization initiation sitehaving a terminal 3′ OH group and with (iii) at least one type of ahexaphosphate nucleotide linked to an energy transfer acceptor moiety,so as to incorporate the hexaphosphate nucleotide onto the 3′OH group.

Provided herein are methods for successively incorporating nucleotidescomprising the steps of: contacting (i) a polymerase according to anyone of SEQ ID NO:1-3 and linked to an energy transfer donor moiety with(ii) a nucleic acid molecule and with (iii) a plurality of more than onetype of a hexaphosphate nucleotide each type of hexaphosphate nucleotidelinked to a different type of energy transfer acceptor moiety, so as tosuccessively incorporate the hexaphosphate nucleotides into the nucleicacid molecule.

Provided herein are methods for conducting a plurality of nucleotideincorporation reactions, each nucleotide incorporation reactioncomprises the steps of: contacting (i) a polymerase according to any oneof SEQ ID NO:1-3 and linked to an energy transfer donor moiety with (ii)a nucleic acid molecule and with (iii) at least one type of ahexaphosphate nucleotide linked to an energy transfer acceptor moiety,so as to incorporate the hexaphosphate nucleotide into the nucleic acidmolecule.

Provided herein are methods for successively incorporating nucleotidescomprising the steps of: contacting (i) a polymerase according to anyone of SEQ ID NO:1-3 and linked to an energy transfer donor nanoparticlewith (ii) a nucleic acid molecule and with (iii) a plurality of morethan one type of a hexaphosphate nucleotide each type of hexaphosphatenucleotide linked to a different type of energy transfer acceptormoiety, so as to successively incorporate the hexaphosphate nucleotidesinto the nucleic acid molecule.

Provided herein are methods for conducting a plurality of nucleotideincorporation reactions, each nucleotide incorporation reactioncomprises the steps of: contacting (i) a polymerase according to any oneof SEQ ID NO:1-3 and linked to an energy transfer donor nanoparticlewith (ii) a nucleic acid molecule and with (iii) at least one type of ahexaphosphate nucleotide linked to an energy transfer acceptor moiety,so as to incorporate the hexaphosphate nucleotide into the nucleic acidmolecule.

Detecting Nucleotide Incorporation

In one embodiment, additional steps can be conducted to detectnucleotide incorporation. The additional steps comprise: (a) excitingthe energy transfer donor moiety with an excitation source; and (b)detecting the energy transfer signal or a change in the energy transfersignal from the incorporated nucleotide whereby the energy transferdonor moiety and the energy transfer acceptor moiety are located inclose proximity to each other.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be light. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal can be a FRET signal. In yet another embodiment, theenergy transfer signal or the change in the energy transfer signal canbe spectrally or optically detectable.

Identifying the Incorporated Nucleotide

In another embodiment, additional steps can be conducted to identify theincorporated nucleotide. The additional steps comprise: (a) exciting theenergy transfer donor moiety with an excitation source; (b) detectingthe energy transfer signal or a change in the energy transfer signalfrom the incorporated nucleotide whereby the energy transfer donormoiety and the energy transfer acceptor moiety are located in closeproximity to each other; and (c) identifying the energy transfer signalor the change in the energy transfer signal from the incorporatednucleotide.

In one embodiment, the excitation source can be electromagnetic energy.In another embodiment, the excitation source can be light. In anotherembodiment, the energy transfer signal or the change in the energytransfer signal can be a FRET signal. In yet another embodiment, theenergy transfer signal or the change in the energy transfer signal canbe spectrally or optically detectable.

Embodiments of Methods for Incorporating Nucleotides

In methods for generating an energy transfer signal, in one embodiment,the energy transfer donor and acceptor moieties can fluoresce inresponse to exposure to an excitation source, such as electromagneticradiation. These fluorescence responses can be an energy transfersignal. In another embodiment, the energy transfer acceptor moiety canfluoresce in response to energy transferred from a proximal excitedenergy transfer donor moiety. These fluorescence responses can be anenergy transfer signal. The proximal distance between the donor andacceptor moieties that accommodates energy transfer can be dependentupon the particular donor/acceptor pair. The proximal distance betweenthe donor and acceptor moieties can be about 1-20 nm, or about 1-10 nm,or about 1-5 nm, or about 5-10 nm. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety can remain unchanged. In another embodiment, the energytransfer signal generated by proximity of the donor moiety to theacceptor moiety results in changes in the energy transfer signal. Inanother embodiment, the changes in the energy transfer signals from thedonor or acceptor moiety can include changes in the: intensity of thesignal; duration of the signal; wavelength of the signal; amplitude ofthe signal; polarization state of the signal; duration between thesignals; and/or rate of the change in intensity, duration, wavelength oramplitude. In another embodiment, the change in the energy transfersignal can include a change in the ratio of the change of the energytransfer donor signal relative to change of the energy transfer acceptorsignals. In another embodiment, the energy transfer signal from thedonor can increase or decrease. In another embodiment, the energytransfer signal from the acceptor can increase or decrease. In anotherembodiment, the energy transfer signal associated with nucleotideincorporation includes: a decrease in the donor signal when the donor isproximal to the acceptor; an increase in the acceptor signal when theacceptor is proximal to the donor; an increase in the donor signal whenthe distance between the donor and acceptor increases; and/or a decreasein the acceptor signal when the distance between the donor and acceptorincreases.

In one embodiment, the detecting the energy transfer signal can beperformed using confocal laser scanning microscopy, Total InternalReflection (TIR), Total Internal Reflection Fluorescence (TIRF),near-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, and/or multi-foci multi-photon.

In practicing the nucleotide binding and/or nucleotide incorporationmethods, non-desirable fluorescent signals can come from sourcesincluding background and/or noise. In one embodiment, the energytransfer signals can be distinguished from the non-desirable fluorescentsignals by measuring, analyzing and characterizing attributes of allfluorescent signals generated by the nucleotide incorporation reaction.In one embodiment, attributes of the energy transfer signal that canpermit distinction from the non-desirable fluorescent signals caninclude: duration; wavelength; amplitude; photon count; and/or the rateof change of the duration, wavelength, amplitude; and/or photon count.In one embodiment, the identifying the energy transfer signal, includesmeasuring, analyzing and characterizing attributes of: duration;wavelength; amplitude; photon count and/or the rate of change of theduration, wavelength, amplitude; and/or photon count. In one embodiment,identifying the energy transfer signal can be used to identify theincorporated nucleotide.

In one embodiment, the nucleic acid molecule can be DNA, RNA or DNA/RNA.

In one embodiment, the polymerase has an active site. The nucleotide canbind the active site. In another embodiment, the polymerase can be aDNA-dependent or RNA-dependent polymerase, or a reverse transcriptase.In another embodiment, the polymerase having altered nucleotide bindingand/or nucleotide incorporation kinetics can improve distinction betweenproductive and non-productive binding events. In another embodiment, thealtered nucleotide binding kinetics and/or altered nucleotideincorporation kinetics can include altered kinetics for: polymerasebinding to the target molecule; polymerase binding to the nucleotide;polymerase catalyzing nucleotide incorporation; the polymerase cleavingthe nucleotide and forming a cleavage product; and/or the polymerasereleasing the cleavage product. In another embodiment, the polymerasecan be linked to an energy transfer donor moiety to form a conjugate. Inanother embodiment, the polymerase component of the conjugate can beenzymatically active. In another embodiment, the polymerase has alteredkinetics for nucleotide binding and/or nucleotide incorporation used incombination with labeled nucleotides having six or more phosphate groups(or substituted phosphate groups), which improve distinction betweenproductive and non-productive binding events. In another embodiment, thepolymerase can have improved photo-stability. The polymerase can be aPhi29-like polymerase, including Phi29 or B103 polymerase. Thepolymerase can be a mutant polymerase. The polymerase can be a B103polymerase according to any one of SEQ ID NOS: 1-5.

In one embodiment, the energy transfer donor moiety can be ananoparticle or a fluorescent dye. The nanoparticle can be about 1-20 nmin its largest dimensions. The nanoparticle can be a core/shellnanoparticle. The nanoparticle can include a core comprisingsemiconductor material(s). The core can include materials (includingbinary, ternary and quaternary mixtures thereof), from: Groups II-VI ofthe periodic table, including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,HgSe, HgTe, MgTe; Groups III-V, including GaN, GaP, GaAs, GaSb, InN,InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/or Group IV, including Ge,Si, Pb. The nanoparticle can include at least one shell surrounding thecore. The shell can include semiconductor material(s). The nanoparticlecan include an inner shell and an outer shell. The shell can includematerials (including binary, ternary and quaternary mixtures thereof)comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs,GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs,AlN, AlP, or AlSb. In one embodiment, the nanoparticle comprises a corehaving CdSe. In another embodiment, the nanoparticle comprises an innershell having CdS. In another embodiment, the nanoparticle comprises anouter shell having ZnS. The outermost surface of the core or shell canbe coated with tightly associated ligands which are not removed byordinary solvation. In some embodiments, the nanoparticle can have alayer of ligands on its surface which can further be cross-linked toeach other. In some embodiments, the nanoparticle can have other oradditional surface coatings which can modify the properties of theparticle, for example, increasing or decreasing solubility in water orother solvents. The nanoparticle can be water dispersible. Thenanoparticle can be a non-blinking nanoparticle. The nanoparticle can bephoto-stable. The nanoparticle may not interfere with polymeraseactivity, including polymerase binding to the target molecule,polymerase binding to the nucleotide, polymerase catalyzing nucleotideincorporation, or the polymerase cleaving the nucleotide and/orreleasing the cleavage product.

In one embodiment, the target nucleic acid molecule can be DNA or RNA orDNA/RNA molecule. In another embodiment, the target nucleic acidmolecule is a single nucleic acid molecule. In another embodiment, thetarget nucleic acid molecule (e.g., target molecule) is base-paired witha polymerization initiation site. In another embodiment, thepolymerization initiation site is a terminal 3′OH of a primer moleculeor of a self-primed target molecule. In another embodiment, thepolymerization initiation site is a 3′OH within a gap or nick. Inanother embodiment, the target nucleic acid molecule and/or thepolymerization initiation site is immobilized to a solid surface. Inanother embodiment, the target nucleic acid molecule is a linear orcircular nucleic acid molecule.

In one embodiment, the at least one type of nucleotide can include 3-10phosphate groups or substituted phosphate groups, or a combination ofphosphate groups and substituted phosphate groups. The nucleotide caninclude a terminal phosphate group or terminal substituted phosphategroup which can be linked to the energy transfer acceptor moiety. Thenucleotide can include the energy transfer acceptor moiety which islinked the base, sugar, or any phosphate group or substituted phosphategroup. The nucleotide can be adenosine, guanosine, cytosine, thymidine,uridine, or any other type of nucleotide.

In one embodiment, the energy transfer acceptor moiety can be afluorescent dye. The energy transfer acceptor moiety and the energytransfer donor moiety can be capable of energy transfer.

In one embodiment, more than one type of nucleotide can be contactedwith the polymerase. Each of the different types of nucleotides can belinked to the same or to different types of energy transfer acceptormoieties, or any combination of the same or different types of acceptormoieties.

In another embodiment, a plurality of one or more different types ofnucleotides can be included in the nucleotide incorporation reaction topermit successive nucleotide incorporation.

Compositions and Systems

Provided herein are compositions and systems, comprising a DNA-dependentpolymerase having properties which offer advantages over otherDNA-dependent polymerase which are traditionally used for nucleotidepolymerization reactions.

For example, the compositions and systems comprise a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation for improved distinction between productive andnon-productive nucleotide binding events. In another example, thecompositions and systems comprise a DNA-dependent polymerase which canpolymerize nucleotides having 4, 5, 6, or more phosphate groups. Inanother example, the compositions and systems comprise a DNA-dependentpolymerase having improved photo-stability when exposed toelectromagnetic energy (e.g., exposed to light during the nucleotideincorporation reactions). In another example, the compositions andsystems comprise a DNA-dependent polymerase which is enzymaticallystable and retains enzymatic activity when linked to an energy transfermoiety. In yet another example, the compositions and systems comprise aDNA-dependent polymerase according to SEQ ID NOS: 1, 2 or 3.

The compositions comprise an energy transfer moiety linked to aDNA-dependent polymerase having altered kinetics for nucleotide bindingand/or nucleotide incorporation for improved distinction betweenproductive and non-productive nucleotide binding events. For example,the compositions comprise a nanoparticle or fluorescent dye linked toDNA-dependent polymerase having altered kinetics for nucleotide bindingand/or nucleotide incorporation. In yet another example, thecompositions comprise an energy transfer donor (e.g., nanoparticle orfluorescent dye) linked to DNA-dependent polymerase according to SEQ IDNOS: 1, 2 or 3.

The compositions comprise an energy transfer moiety linked to aDNA-dependent polymerase having altered kinetics for nucleotide bindingand/or nucleotide incorporation and the polymerase is bound to a targetnucleic acid molecule. In one embodiment, the compositions comprises anenergy transfer moiety linked to a DNA-dependent polymerase havingaltered kinetics for nucleotide binding and/or nucleotide incorporationand the polymerase is bound to a target nucleic acid molecule which isbase-paired with a polymerization initiation site having a 3′OH group.In another embodiment, the compositions comprises an energy transfermoiety linked to a DNA-dependent polymerase according to SEQ ID NOS: 1,2 or 3, and the polymerase is bound to a target nucleic acid molecule.In another embodiment, the compositions comprises an energy transfermoiety linked to a DNA-dependent polymerase according to SEQ ID NOS: 1,2 or 3, and the polymerase is bound to a target nucleic acid moleculewhich is base-paired with a polymerization initiation site having a 3′OHgroup.

Embodiments of the compositions include: the target molecule can bebase-paired with a polymerization initiation site having a 3′ OH group;the target molecule can be base-paired with a nucleic acid primer; thetarget molecule can be immobilized; the nucleic acid primer molecule canbe immobilized; and/or the target and primer molecules can beimmobilized.

Provided herein are systems, comprising a DNA-dependent polymerasehaving properties which offer advantages over other DNA-dependentpolymerase which are traditionally used for nucleotide polymerizationreactions.

For example, the systems comprise an energy transfer moiety linked to aDNA-dependent polymerase having altered kinetics for nucleotide bindingand/or nucleotide incorporation.

In another example, the systems comprise a nanoparticle or fluorescentdye linked to a DNA-dependent polymerase having altered kinetics fornucleotide binding and/or nucleotide incorporation.

In another example, the systems comprise an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule.

In another example, the systems comprise an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule which is base-paired with a polymerization initiation sitehaving a 3′OH group.

In another example, the systems comprise an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule which is base-paired with a nucleic acid primer molecule.

In another example, the systems comprise (i) an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule and (ii) a nucleotide linked to an energy transfer moiety.

In another example, the systems comprise (i) an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule which is base-paired with a polymerization initiation sitehaving a 3′OH group and (ii) a nucleotide linked to an energy transfermoiety.

In another example, the systems comprise (i) an energy transfer moiety(e.g., nanoparticle or fluorescent dye) linked to a DNA-dependentpolymerase having altered kinetics for nucleotide binding and/ornucleotide incorporation and the polymerase is bound to a target nucleicacid molecule which is base-paired with a nucleic acid primer and (ii) anucleotide linked to an energy transfer moiety.

Embodiments of the systems include: the DNA-dependent polymerase havingaltered kinetics for nucleotide binding and/or nucleotide incorporationcan be any of SEQ ID NOS:1, 2, or 3; the target molecule can bebase-paired with a polymerization initiation site having a 3′ OH group;the target molecule can be base-paired with a nucleic acid primer; thetarget molecule can be immobilized; the nucleic acid primer molecule canbe immobilized; the target and primer molecules can be immobilized; theenergy transfer moiety which is linked to the nucleotide can be anenergy transfer acceptor moiety (e.g., fluorescent dye).

Reagent Exchange Methods

Provided herein are compositions, systems, methods, and kits, forexchanging (e.g., replacing) the reagents for nucleotide binding ornucleotide incorporation reactions with fresh reagents on the sametarget nucleic acid molecule.

The methods for exchanging the reagents can be used for: re-sequencingat least a portion of the same nucleic acid molecule; or replacing anyreagent used to practice nucleotide binding and/or nucleotideincorporation with functional reagents to permit continuing thenucleotide incorporation reaction on the same nucleic acid molecule; orperforming nucleotide binding or nucleotide incorporation reactions andswitching to reactions having different nucleotide binding and/ornucleotide incorporation reaction properties on the same nucleic acidmolecule.

The reagents which can be exchanged include any reagent which is used ina nucleotide binding or nucleotide incorporation reaction, including butnot limited to any type of: target molecule; primer; polymerizationinitiation site; polymerase; nucleotides (e.g., hydrolyzable,non-hydrolyzable, chain-terminating, or labeled or non-labelednucleotides); the synthesized strand; compounds which reducephoto-damage; buffers; salts; co-factors; divalent cations; andchelating agents. The fresh reagents can be the same or different typesof reagents compared to the old reagents.

For example, one round of a reagent exchange reaction can be conductedusing three types of nucleotides (e.g., A, G, and C) labeled with adifferent type of energy transfer acceptor dye, and another differenttype of nucleotide (e.g., T) can be unlabeled. In one embodiment, the Anucleotides can be labeled with dye type 1, G nucleotides can be labeledwith dye type 2, and C nucleotides can be labeled with dye type 3. In asecond round, the reagent exchange reaction can be conducted using threetypes of nucleotides (e.g., G, C, and T) labeled with a different typeof energy transfer acceptor dye, and another different type ofnucleotide (e.g., A) can be unlabeled. In a third round, the reagentexchange reaction can be conducted using three types of nucleotides(e.g., C, T, and A) labeled with a different type of energy transferacceptor dye, and another different type of nucleotide (e.g., G) can beunlabeled. In a fourth round, the reagent exchange reaction can beconducted using three types of nucleotides (e.g., T, A, and G) labeledwith a different type of energy transfer acceptor dye, and anotherdifferent type of nucleotide (e.g., C) can be unlabeled. The first,second, third, and fourth rounds of reagent exchange reactions can beconducted in any order, and in any combination. In any of the rounds ofreagent exchange reactions, the different types of nucleotides can belinked to the same or different type of energy transfer dye.

In another example, multiple rounds of reagent exchange reactions can beconducted using four types of nucleotides (e.g., A, G, C, and T) eachlabeled with a different type of energy transfer acceptor dye in eachround. In one embodiment, in round one, the A nucleotides can be labeledwith dye type 1, G labeled with dye type 2, C labeled with dye type 3,and T labeled with dye type 4. In a subsequent round, the reagentexchange reaction can be conducted using A labeled with dye type 2, Glabeled with dye type 3, C labeled with dye type 4, and T labeled withdye type 1. One skilled in the art will readily recognize that manycombinations are possible.

In one aspect, the reagent exchange methods can be used to sequence thesame target nucleic acid molecule 1, 2, 3, 4, or 5 times, or up to 10times, or up to 25 times, or up to 50 times, or more than 50 times. Forexample, errors in detecting and/or identifying the incorporatednucleotides may necessitate re-sequencing the same target molecule. Theerrors can arise when a non-reporting nucleotide (e.g., which is linkedto a non-reporting energy transfer acceptor dye) is incorporated butdoes not emit a detectable signal. The same target molecule can besequenced one or more times to provide redundant nucleotide sequenceinformation. The reagent exchange methods can be used to sequence thestrand which is synthesized during a nucleotide incorporation reaction.The synthesized strand can be sequenced 1, 2, 3, 4, or 5 times, or up to10 times, or up to 25 times, or up to 50 times, or more than 50 times,to provide redundant nucleotide sequence information. The same targetmolecule, or synthesized strand, can be re-sequenced using exchangedprimers having sequences which are the same or a different from thesequence of the old primers. Sequencing the same target moleculemultiple times, and/or sequencing the same synthesized strand multipletimes, can provide multiple data sets of sequence information which canbe aligned and compared. In one embodiment, the alignment can be used todeduce a consensus sequence of the target molecule or the synthesizedstrand. The alignment can be used to provide multi-fold coverage of thenucleotides which are contained within the target molecule orsynthesized strand.

The reagent exchange methods can be used to replace inactive polymerasesand/or non-functional nucleotides or energy transfer moieties, withfresh polymerase, nucleotides, and/or other reagents, in order tocontinue the nucleotide incorporation reaction on the same targetmolecule or synthesized strand. For example, fresh polymerase,nucleotides, and/or reagents can be added to the immobilizedtarget/primer molecules to permit continuation of the nucleotideincorporation reaction on the same target or synthesized molecule.

The reagent exchange methods can be used to replace the reagents in anon-going nucleotide binding or incorporation reaction, in order toswitch to a different type of nucleotide binding or nucleotideincorporation reaction on the same target or synthesized molecule. Forexample, the first nucleotide incorporation reaction can be conductedusing a polymerase, nucleotides, and other reagents, which exhibitcertain properties, such as: nucleotide fidelity; rate of nucleotideincorporation; processivity; strand displacement; kinetics of nucleotidebinding, catalysis, release of the cleavage product, and/or polymerasetranslocation; exonuclease activity; and/or activity at certaintemperatures. The reagents (e.g., polymerases and/or nucleotides) can beexchanged with different reagents to conduct a nucleotide incorporationreaction which exhibits different nucleotide incorporation properties(on the same target molecule or on the same synthesized strand).

The reagent exchange methods can be practiced using any type ofnucleotide binding or nucleotide incorporation reactions, including butnot limited to: the energy transfer methods disclosed herein; any typeof discontinuous reactions (e.g., synchronous nucleotide incorporationmethods described in: (U.S. Ser. No. 61/184,774, filed on Jun. 5, 2009;U.S. Ser. No. 61/242,762, filed on Sep. 15, 2009; and U.S. Ser. No.61/180,811, filed on May 22, 2009; U.S. Ser. No. 61/295,533, filed onJan. 15, 2010); and any type of continuous reactions (e.g., asynchronousnucleotide incorporation methods as described in: (U.S. Ser. No.61/077,090, filed on Jun. 30, 2008; U.S. Ser. No. 61/089,497, filed onAug. 15, 2008; U.S. Ser. No. 61/090,346, filed on Aug. 20, 2008; PCTapplication No. PCT/US09/049324, filed on Jun. 30, 2009; U.S. Ser. No.61/164,324, filed on Mar. 27, 2009; and U.S. Ser. No. 61/263,974, filedon Nov. 24, 2009; U.S. Ser. Nos. 61/289,388; 61/293,616; 61/299,917;61/307,356).

The reagent exchange methods can be practiced using any type of formatusing an immobilized: primer; target molecule; synthesized strand;and/or polymerase. The reagent exchange methods can be practiced on asingle target nucleic acid molecule, or on random or organized arrays ofsingle nucleic acid molecules, and using any type of solid surface (U.S.Ser. No. 61/220,174, filed on Jun. 24, 2009; and U.S. Ser. No.61/245,248, filed on Sep. 23, 2009; U.S. Ser. No. 61/302,475). Thetarget molecules and synthesized strands can be genomic, recombinant,DNA, RNA, double-stranded, or single-stranded nucleic acid molecules.The target nucleic acid molecules can be linear or circular. The targetnucleic acid molecules can be self-priming molecules or can beassociated with primer molecules. The target nucleic acid molecules canbe immobilized using any method, including the methods depicted in anyof FIGS. 2-8.

Provided herein are reagent exchange methods, where the existing targetmolecule, synthesized strand, primer, polymerase, nucleotides, and/orother reagents, can be removed in a manner which does not remove theimmobilized target molecule, primer, or synthesized strand. In someembodiments, the primer, target molecule, or synthesized strand can beremoved. Methods for removing the components include physical, chemical,and/or enzymatic methods.

The polymerase can be inactivated and/or removed using physical,chemical, and/or enzymatic method, in any combination and in any order.For example, the polymerase can be deactivated using elevatedtemperatures, such as 45-80° C., for about 30 seconds to 10 minutes. Inanother example, the polymerase can be removed from the target moleculeor synthesized strand using a protein-degrading enzyme, such asproteinase-K. In another example, the polymerase can be removed from thetarget molecule or synthesized strand using compounds known to disruptprotein complexes, where the compounds include detergents (e.g.,N-lauroyl sarcosine, SDS), chaotropic salt (e.g., guanidiniumhydrochloride), lithium sulfate, and EDTA.

Any combination of capture molecule, primer, target molecule, and/orsynthesized strand, can be dissociated (e.g., denatured) from each otherusing physical, chemical, and/or enzymatic methods, in any combinationand in any order. For example, the target molecule/synthesized strandduplex can be denatured using elevated temperatures, such as about75-100° C. (e.g., without formamide) or about 45-90° C. (e.g., withformamide). In another example, the target molecule or synthesizedstrand can be degraded using a nucleic acid degrading enzyme, such as a5′→3′ or 3′→5′ exonuclease (e.g., exonuclease III, T7 gene 6exonuclease, exonuclease I). In yet another example, the target moleculeor synthesized strand can be denatured using any compound known todissociate double-stranded nucleic acid molecules, such as anycombination of: formamide, urea, DMSO, alkali conditions (e.g., NaOH atabout 0.01-0.3 M, or about 0.05-0.1 M; e.g., elevated pH of about 7-12),or low salt or very-low salt conditions (e.g., about less than 0.001-0.3mM cationic conditions), or water.

In practicing the reagent exchange methods, the target molecule,synthesized strand, polymerase, primer, capture molecule, or anyreagent, can be removed using fluid flow, washing, and/or aspiration.The target molecule, primer molecule, synthesized strand, or capturemolecule can be operably linked to the solid surface in a manner whichwithstands flowing, washing, aspirating, and changes in salt,temperature, chemical, enzymatic, and/or pH conditions. A fresh supplyof polymerase, nucleotides, reagents, primer molecules, splintermolecules, and/or adaptor molecules, can be added to the immobilizednucleic acid molecules. The polymerase (e.g., donor-labeled) andnucleotides (e.g., and acceptor-labeled) can be added to the immobilizednucleic acid molecules under conditions which are suitable fornucleotide binding and/or nucleotide incorporation to occur. The freshpolymerase, nucleotides, and reagents, can be the same or different fromthe old polymerase, nucleotides, and/or reagents.

In the following embodiments (e.g., FIGS. 2-8), the “N” can be anynucleotide base, and the “I” can be a universal base such as inosine.

In one embodiment, a target molecule can be ligated to an immobilizedcapture molecule using a splinter oligonucleotide (which can hybridizeto the target molecule and capture oligonucleotide) and enzymes forligation and/or nucleotide polymerization (e.g., T4 ligase and T4 DNApolymerase, respectively) (see FIG. 2). A primer can be annealed to theimmobilized target molecule, and a synthesized strand can be producedusing a polymerase and nucleotides. Physical, chemical, and/or enzymaticconditions can be used to remove the synthesized strand, polymerase, andnucleotides. The remaining target molecule can be contacted with freshreagents to permit re-sequencing the same target molecule. FIG. 2depicts re-sequencing the same target molecule, in a direction away fromthe solid surface. A “two-pass” method for re-sequencing the samenucleic acid molecule has been described (Harris, et al., 2008 Science320:106-109, and supporting online material).

In another embodiment, a polynucleotide tail (e.g., poly-A, -G, -C, or-T) can be added to a target molecule, for example using a terminaltransferase enzyme (TdT in FIG. 3). The tailed target molecule can beligated to an immobilized capture molecule using a splinteroligonucleotide (which can hybridize to the target molecule and captureoligonucleotide) and enzymes for ligation and/or nucleotidepolymerization (e.g., T4 ligase and T4 DNA polymerase, respectively). Aprimer can be annealed to the immobilized target molecule, and asynthesized strand can be produced using a polymerase and nucleotides.Physical, chemical, and/or enzymatic conditions can be used to removethe synthesized strand, polymerase, and nucleotides. The remainingtarget molecule can be contacted with fresh reagents to permitre-sequencing the same target molecule. FIG. 3 depicts re-sequencing thesame target molecule, in a direction away from the solid surface.

In yet another embodiment, a target molecule can be ligated to animmobilized hairpin capture molecule, where a portion of the capturemolecule can hybridize to the target molecule (see FIG. 4). The targetmolecule can be ligated to the hairpin capture molecule using enzymesfor ligation and/or nucleotide polymerization (e.g., T4 ligase and T4DNA polymerase, respectively). The hairpin adaptor molecule can includea recognition sequence for cleavage (scission) by an endonucleaseenzyme. For example, the recognition sequence can be an RNA portionwhich can be 3-6 nt in length, to form a DNA/RNA hybrid. The RNA portioncan be 4 nt in length. The RNA portion can include purines (A and G) inany order. The RNA portion of the RNA/DNA duplex can be a substrate forcleavage by an endoribonuclease (e.g., RNase H). In another example, therecognition sequence can be an AP site (apurinic/apyrimidinic) having aTHF substrate (tetrahydrofuran) which can be cleaved by an APendonuclease. In another example, the recognition sequence can includenucleotide analogs (e.g., 8-oxo-7,8-dihydroguanine, 8-oxoguanine, or8-hydroxyguanine) which can be cleaved by DNA glycosylase OGG1. In yetanother example, the recognition sequence can include any sequence whichcan be cleaved by a nicking enzyme. After scission, a primer can beannealed to the target molecule, and a synthesized strand can beproduced using a polymerase and nucleotides. Physical, chemical, and/orenzymatic conditions can be used to remove the synthesized strand,polymerase, and nucleotides. The remaining target molecule can becontacted with fresh reagents to permit re-sequencing the same targetmolecule. FIG. 4 depicts re-sequencing the same target molecule, in adirection away from the solid surface.

In yet another embodiment, the 5′ end of a target molecule can beligated to an adaptor molecule using T4 ligase (FIG. 5A). The adaptormolecule can be annealed with a primer having a blocked 3′ end (FIG.5A). The target molecule can be reacted with terminal transferase to adda poly-nucleotide tail (e.g., poly-A, -G, -C, or -T) (TdT in FIG. 5A).The tailed target molecule can be captured by an immobilizedoligonucleotide (FIG. 5B). The immobilized oligonucleotide can be usedto produce a synthesized strand, using a polymerase and nucleotides(FIG. 5B). Physical, chemical, and/or enzymatic conditions can be usedto remove the target strand, polymerase, and nucleotides. A primer canbe annealed to the remaining synthesized strand. A newly synthesizedstrand can be produced using a polymerase and nucleotides. Physical,chemical, and/or enzymatic conditions can be used to remove the newlysynthesized strand, polymerase, and nucleotides. The remainingsynthesized strand can be contacted with fresh reagents to permitre-sequencing the same synthesized strand. FIGS. 5A and B depictre-sequencing the same synthesized strand, in a direction towards thesolid surface.

In yet another embodiment, the target molecule can be reacted withterminal transferase to add a poly-nucleotide tail (e.g., poly-A, -G,-C, or -T) (TdT in FIG. 6A). The tailed target molecule can be capturedby an immobilized capture oligonucleotide (FIG. 6A). The immobilizedcapture oligonucleotide can be used to generate a synthesized strand,using a polymerase and nucleotides (FIG. 6B). The 3′ end of thesynthesized strand can be ligated to an adaptor molecule. Physical,chemical, and/or enzymatic conditions can be used to remove the targetmolecule, polymerase, and nucleotides. The 3′ end of the remainingsynthesized strand can be annealed to a primer. A newly synthesizedstrand can be generated with a polymerase and nucleotides. Physical,chemical, and/or enzymatic conditions can be used to remove the newlysynthesized strand, polymerase, and nucleotides. The remainingsynthesized strand can be contacted with fresh reagents to permitre-sequencing the same synthesized strand. FIGS. 6A and B depictre-sequencing the same synthesized strand, in a direction towards thesolid surface.

In yet another embodiment, the target molecule can be reacted withterminal transferase to add a poly-nucleotide tail (e.g., poly-A, -G,-C, or -T) (TdT in FIG. 7). The tailed target molecule can becircularized. The circularized target molecule can be captured by animmobilized oligonucleotide. The 3′ end of the capture oligonucleotidecan be used to generate a synthesized strand using a polymerase andnucleotides, in a rolling circle replication mode. A strand-displacementDNA polymerase can be used for the rolling circle replication.

In another embodiment, stem-loop adaptor molecules can be ligated toboth ends of a double-stranded target molecule using T4 ligase (FIG. 8)to produce a closed-ended molecule. The resulting molecule can becaptured by an immobilized oligonucleotide via complementary sequencesin one of the stem-loop adaptor molecules. The immobilized captureoligonucleotide can be used as a primer to generate the synthesizedstrand, using a polymerase and nucleotides.

Nucleotides

The methods, compositions, systems and kits disclosed herein can includenucleotides. The nucleotides can be linked with at least one energytransfer moiety (FIG. 1). The energy transfer moiety can be an energytransfer acceptor or donor moiety. The different types of nucleotides(e.g., adenosine, thymidine, cytidine, guanosine, and uridine) can belabeled with a different type energy transfer acceptor or donor moietyso that the detectable signals (e.g., energy transfer signals) from eachof the different types nucleotides can be distinguishable to permit baseidentity. In one embodiment, the different types of nucleotides (e.g.,adenosine, thymidine, cytidine, guanosine, and uridine) can be labeledwith a different type of energy transfer acceptor moiety so that thedetectable signals (e.g., energy transfer signals) from each of thedifferent types nucleotides can be distinguishable to permit baseidentity. The nucleotides can be labeled in a way that does notinterfere with the events of nucleotide polymerization. For example theattached energy transfer acceptor moiety does not interfere with:nucleotide binding; nucleotide incorporation; cleavage of thenucleotide; or release of the cleavage product. See for example, U.S.Ser. No. 61/164,091, Ronald Graham, concurrently filed Mar. 27, 2009.See for example U.S. Pat. Nos. 7,041,812, 7,052,839, 7,125,671, and7,223,541; U.S. Pub. Nos. 2007/0072196 and 2008/0091005; Sood et al.,2005, J. Am. Chem. Soc. 127:2394-2395; Arzumanov et al., 1996, J. Biol.Chem. 271:24389-24394; and Kumar et al., 2005, Nucleosides, Nucleotides& Nucleic Acids, 24(5):401-408.

In one aspect, the energy transfer acceptor moiety may be linked to anyposition of the nucleotide. For example, the energy transfer acceptormoiety can be linked to any phosphate group (or substituted phosphategroup), the sugar or the base. In another example, the energy transfermoiety can be linked to any phosphate group (or substituted phosphategroup) which is released as part of a phosphate cleavage product uponincorporation. In yet another example, the energy transfer acceptormoiety can be linked to the terminal phosphate group (or substitutedphosphate group). In another aspect, the nucleotide may be linked withan additional energy transfer acceptor moiety, so that the nucleotide isattached with two or more energy transfer acceptor moieties. Theadditional energy transfer acceptor moiety can be the same or differentas the first energy transfer acceptor moiety. In one embodiment, theenergy transfer acceptor moiety can be a FRET acceptor moiety.

In one aspect, the nucleotide may be linked with a reporter moiety whichis not an energy transfer moiety. For example, the reporter moiety canbe a fluorophore.

In one aspect, the energy transfer acceptor moieties and/or the reportermoiety can be attached to the nucleotide via a linear or branched linkermoiety. An intervening linker moiety can connect the energy transferacceptor moieties with each other and/or to the reporter moiety, in anycombination of linking arrangements.

In another aspect, the nucleotides comprise a sugar moiety, base moiety,and at least three, four, five, six, seven, eight, nine, ten, or morephosphate groups (or substituted phosphate groups) linked to the sugarmoiety by an ester or phosphoramide linkage. The phosphates can belinked to the 3′ or 5′ C of the sugar moiety.

In one aspect, different linkers can be used to operably link thedifferent nucleotides (e.g., A, G, C, T or U) to the energy transfermoieties or reporter moieties. For example, adenosine nucleotide can beattached to one type of energy transfer moiety using one type of linker,and guanosine nucleotide can be linked to a different type of energytransfer moiety using a different type of linker. In another example,adenosine nucleotide can be attached to one type of energy transfermoiety using one type of linker, and the other types of nucleotides canbe attached to different types of energy transfer moieties using thesame type of linker. One skilled in the art will appreciate that manydifferent combinations of nucleotides, energy transfer moieties, andlinkers are possible.

In one aspect, the distance between the nucleotide and the energytransfer moiety can be altered. For example, the linker length and/ornumber of phosphate groups (or substitute phosphate groups) can lengthenor shorten the distance from the sugar moiety to the energy transfermoiety. In another example, the distance between the nucleotide and theenergy transfer moiety can differ for each type of nucleotide (e.g., A,G, C, T or U).

In another aspect, the number of energy transfer moieties which arelinked to the different types of nucleotides (e.g., A, G, C, T or U) canbe the same or different. For example: A can have one dye, and G, C, andT have two; A can have one dye, C has two, G has three, and T has four;A can have one dye, C and G have two, and T has four. One skilled in theart will recognize that many different combinations are possible.

In another aspect, the concentration of the labeled nucleotides used toconduct the nucleotide binding or nucleotide incorporation reactions, orthe concentration included in the systems or kits, can be about 0.0001nM-1 μM, or about 0.0001 nM-0.001 nM, or about 0.001 nM-0.01 nM, orabout 0.01 nM-0.1 nM, or about 0.1 nM-1.0 nM, or about 1 nM-25 nM, orabout 25 nM-50 nM, or about 50 nM-75 nM, or about 75 nM-100 nM, or about100 nM-200 nM, or about 200 nM-500 nM, or about 500 nM-750 nM, or about750 nM-1000 nM, or about 0.1 μM-20 μM, or about 20 μM-50 μM, or about 50μM-75 μM, or about 75 μM-100 μM, or about 100 μM-200 μM, or about 200μM-500 μM, or about 500 μM-750 μM, or about 750 μM-1000 μM.

In another aspect, the concentration of the different types of labelednucleotides, which are used to conduct the nucleotide binding orincorporation reaction, can be the same or different from each other.

Sugar Moieties

The nucleotides typically comprise suitable sugar moieties, such ascarbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48),acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal ChemistryLetters vol. 7: 3013-3016), and other suitable sugar moieties (Joeng, etal., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem.36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No.5,558,991). The sugar moiety may be selected from the following:ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl,2′,3′-didehydrodideoxyribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl,2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoriboxyl,2′-alkylthioribosyl, 3′-alkoxyribosyl, 3′-azidoribosyl, 3′-aminoribosyl,3′-fluororibosyl, 3′-mercaptoriboxyl, 3′-alkylthioribosyl carbocyclic,acyclic and other modified sugars. In one aspect, the 3′-position has ahydroxyl group, for strand/chain elongation.

Base Moieties

The nucleotides typically comprise a hetero cyclic base which includessubstituted or unsubstituted nitrogen-containing parent heteroaromaticring which is commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants. Thebase is capable of forming Watson-Crick and/or Hoogstein hydrogen bondswith an appropriate complementary base. Exemplary bases include, but arenot limited to, purines and pyrimidines such as: 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-Δ²-isopentenyladenine(6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine,guanine (G), isoguanine, N²-dimethylguanine (dmG), 7-methylguanine(7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine andO⁶-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),5,6-dihydrothymine, 0⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines;hydroxymethylcytosines; 5-methycytosines; base (Y); as well asmethylated, glycosylated, and acylated base moieties; and the like.Additional exemplary bases can be found in Fasman, 1989, in: PracticalHandbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press,Boca Raton, Fla., and the references cited therein.

Phosphate Groups

The nucleotides typically comprise phosphate groups which can be linkedto the 2′, 3′ and/or 5′ position of the sugar moiety. The phosphategroups include analogs, such as phosphoramidate, phosphorothioate,phosphorodithioate, and O-methylphosphoroamidite groups. In oneembodiment, at least one of the phosphate groups can be substituted witha fluoro and/or chloro group. The phosphate groups can be linked to thesugar moiety by an ester or phosphoramide linkage. Typically, thenucleotide comprises three, four, five, six, seven, eight, nine, ten, ormore phosphate groups linked to the 5′ position of the sugar moiety.

Non-Hydrolyzable Nucleotides

The methods, compositions, systems and kits disclosed herein can includenon-hydrolyzable nucleotides. The nucleotide binding and nucleotideincorporation methods can be practiced using incorporatable nucleotidesand non-hydrolyzable nucleotides. In the presence of the incorporatablenucleotides (e.g., labeled), the non-hydrolyzable nucleotides (e.g.,non-labeled) can compete for the polymerase binding site to permitdistinction between the complementary and non-complementary nucleotides,or for distinguishing between productive and non-productive bindingevents. In the nucleotide incorporation reaction, the presence of thenon-hydrolyzable nucleotides can alter the length of time, frequency,and/or duration of the binding of the labeled incorporatable nucleotidesto the polymerase.

The non-hydrolyzable nucleotides can be non-labeled or can be linked toa reporter moiety (e.g., energy transfer moiety). The labelednon-hydrolyzable nucleotides can be linked to a reporter moiety at anyposition, such as the sugar, base, or any phosphate (or substitutedphosphate group). For example, the non-hydrolyzable nucleotides can havethe general structure:R₁₁—(—P)_(n)—S—B

Where B can be a base moiety, such as a hetero cyclic base whichincludes substituted or unsubstituted nitrogen-containing heteroaromaticring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, orglucosyl group. Where n can be 1-10, or more. Where P can be one or moresubstituted or unsubstituted phosphate or phosphonate groups. Where R₁₁,if included, can be a reporter moiety (e.g., a fluorescent dye). In oneembodiment, the non-hydrolyzable nucleotide having multiple phosphate orphosphonate groups, the linkage between the phosphate or phosphonategroups can be non-hydrolyzable by the polymerase. The non-hydrolyzablelinkages include, but are not limited to, amino, alkyl, methyl, and thiogroups. The phosphate or phosphonate portion of the non-hydrolyzablenucleotide can have the general structure:

Where B can be a base moiety and S can be a sugar moiety. Where any oneof the R₁-R₇ groups can render the nucleotide non-hydrolyzable by apolymerase. Where the sugar C5 position can be CH₂, CH₂O, CH═, CHR, orCH₂ CH₂. Where the R₁ group can be O, S, CH═, CH(CN), or NH. Where theR₂, R₃, and R₄, groups can independently be O, BH₃, or SH. Where the R₅and R₆ groups can independently be an amino, alkyl, methyl, thio group,or CHF, CF₂, CHBr, CCl₂, O—O, or —C≡C—. Where the R₇ group can beoxygen, or one or more additional phosphate or phosphonate groups, orcan be a reporter moiety. Where R₈ can be SH, BH₃, CH₃, NH₂, or a phenylgroup or phenyl ring. Where R₉ can be SH. Where R₁₀ can be CH₃,N₃CH₂CH₂, NH₂, ANS, N₃, MeO, SH, Ph, F, PhNH, PhO, or RS (where Ph canbe a phenyl group or phenyl ring, and F can be a fluorine atom orgroup). The substituted groups can be in the S or R configuration.

The non-hydrolyzable nucleotides can be alpha-phosphate modifiednucleotides, alpha-beta nucleotides, beta-phosphate modifiednucleotides, beta-gamma nucleotides, gamma-phosphate modifiednucleotides, caged nucleotides, or di-nucleotides.

Many examples of non-hydrolyzable nucleotides are known (Rienitz 1985Nucleic Acids Research 13:5685-5695), including commercially-availableones from Jena Bioscience (Jena, Germany).

Polymerases

The compositions, methods, systems and kits disclosed herein involve theuse of one or more polymerases. In some embodiments, the polymeraseincorporates one or more nucleotides into a nucleic acid molecule.

In some embodiments, the polymerase provided herein can offer unexpectedadvantages over polymerases that are traditionally used for nucleotidepolymerization reactions. In some embodiments, the polymerases can beenzymatically active when conjugated to an energy transfer moiety (e.g.,donor moiety). In some embodiments, the polymerases have alteredkinetics for nucleotide binding and/or nucleotide incorporation whichimprove distinction between productive and non-productive nucleotidebinding events. In some embodiments, the polymerases having alteredkinetics for nucleotide binding and/or nucleotide incorporation can beused in combination with labeled nucleotides having six or morephosphate groups (or substituted phosphate groups), which improvesdistinction between productive and non-productive binding events. Insome embodiments, the polymerases have improved photo-stability comparedto polymerases traditionally used for nucleotide polymerization.Examples of polymerases having altered kinetics for nucleotide bindingand/or nucleotide incorporation include B103 polymerases disclosed inU.S. Ser. Nos. 61/242,771, 61/293,618, and any one of SEQ ID NOS: 1-5.

In some embodiments, the polymerase can be unlabeled. Alternatively, thepolymerase can be linked to one or more reporter moiety. In someembodiments, the reporter moiety comprises at least one energy transfermoiety.

The polymerase may be linked with at least one energy transfer donor oracceptor moiety. One or more energy transfer donor or acceptor moietycan be linked to the polymerase at the amino end or carboxyl end or maybe inserted at any site therebetween. Optionally, the energy transferdonor or acceptor moiety can be attached to the polymerase in a mannerwhich does not significantly interfere with the nucleotide bindingactivity, or with the nucleotide incorporation activity of thepolymerase. In such embodiments, the energy transfer donor or acceptormoiety is attached to the polymerase in a manner that does notsignificantly interfere with polymerase activity.

In one aspect, a single energy transfer donor or acceptor moiety can belinked to more than one polymerase and the attachment can be at theamino end or carboxyl end or may be inserted within the polymerase.

In another aspect, a single energy transfer donor or acceptor moiety canbe linked to one polymerase.

In one aspect, the energy transfer donor moiety can be a nanoparticle(e.g., a fluorescent nanoparticle) or a fluorescent dye. The polymerase,which can be linked to the nanoparticle or fluorescent dye, typicallyretains one or more activities that are characteristic of thepolymerase, e.g., polymerase activity, exonuclease activity, nucleotidebinding, and the like.

In one aspect, the polymerases can be replicases, DNA-dependentpolymerases, primases, RNA-dependent polymerases (includingRNA-dependent DNA polymerases such as, for example, reversetranscriptases), strand-displacement polymerases, or thermo-stablepolymerases. In another aspect, the polymerase can be any Family A or Btype polymerase. Many types of Family A (e.g., E. coli Pol I), B (e.g.,E. coli Pol II), C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic PolII), X (e.g., human Pol beta), and Y (e.g., E. coli UmuC/DinB andeukaryotic RAD30/xeroderma pigmentosum variants) polymerases aredescribed in Rothwell and Watsman 2005 Advances in Protein Chemistry71:401-440.

In yet another aspect, the polymerases can be isolated from a cell, orgenerated using recombinant DNA technology or chemical synthesismethods. In another aspect, the polymerases can be expressed inprokaryote, eukaryote, viral, or phage organisms. In another aspect, thepolymerases can be post-translationally modified proteins or fragmentsthereof.

In one aspect, the polymerase can be a recombinant protein which isproduced by a suitable expression vector/host cell system. Thepolymerases can be encoded by suitable recombinant expression vectorscarrying inserted nucleotide sequences of the polymerases. Thepolymerase sequence can be linked to a suitable expression vector. Thepolymerase sequence can be inserted in-frame into the suitableexpression vector. The suitable expression vector can replicate in aphage host, or a prokaryotic or eukaryotic host cell. The suitableexpression vector can replicate autonomously in the host cell, or can beinserted into the host cell's genome and be replicated as part of thehost genome. The suitable expression vector can carry a selectablemarker which confers resistance to drugs (e.g., kanamycin, ampicillin,tetracycline, chloramphenicol, or the like), or confers a nutrientrequirement. The suitable expression vector can have one or morerestriction sites for inserting the nucleic acid molecule of interest.The suitable expression vector can include expression control sequencesfor regulating transcription and/or translation of the encoded sequence.The expression control sequences can include: promoters (e.g., inducibleor constitutive), enhancers, transcription terminators, and secretionsignals. The expression vector can be a plasmid, cosmid, or phagevector. The expression vector can enter a host cell which can replicatethe vector, produce an RNA transcript of the inserted sequence, and/orproduce protein encoded by the inserted sequence. The recombinantpolymerase can include an affinity tag for enrichment or purification,including a poly-amino acid tag (e.g., poly His tag), GST, and/or HAsequence tag. Methods for preparing suitable recombinant expressionvectors and expressing the RNA and/or protein encoded by the insertedsequences are well known (Sambrook et al, Molecular Cloning (1989)).

The polymerases may be DNA polymerases and include without limitationbacterial DNA polymerases, prokaryotic DNA polymerase, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases. The polymerase can be a commercially availablepolymerase.

In some embodiments, the polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

Suitable bacterial DNA polymerase include without limitation E. coli DNApolymerases I, II and III, IV and V, the Klenow fragment of E. coli DNApolymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridiumthermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNApolymerase.

Suitable eukaryotic DNA polymerases include without limitation the DNApolymerases α, δ, ε, η, ζ, γ, β, σ, λ, μ, ι, and κ, as well as the Rev1polymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT).

Suitable viral and/or phage DNA polymerases include without limitationT4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Phi-15 DNApolymerase, Phi-29 DNA polymerase (see, e.g., U.S. Pat. No. 5,198,543;also referred to variously as Φ29 polymerase, phi29 polymerase, phi 29polymerase, Phi 29 polymerase, and Phi29 polymerase); Φ15 polymerase(also referred to herein as Phi-15 polymerase); Φ21 polymerase (Phi-21polymerase); PZA polymerase; PZE polymerase, PRD1 polymerase; Nfpolymerase; M2Y polymerase; SF5 polymerase; f1 DNA polymerase, Cp-1polymerase; Cp-5 polymerase; Cp-7 polymerase; PR4 polymerase; PR5polymerase; PR722 polymerase; L17 polymerase; M13 DNA polymerase, RB69DNA polymerase, G1 polymerase; GA-1 polymerase, BS32 polymerase; B103polymerase; BA103 polymerase, a polymerase obtained from any phi-29 likephage or derivatives thereof, etc. See, e.g., U.S. Pat. No. 5,576,204,filed Feb. 11, 1993; U.S. Pat. Appl. No. 2007/0196846, published Aug.23, 2007.

Suitable archaeal DNA polymerases include without limitation thethermostable and/or thermophilic DNA polymerases such as, for example,DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase,Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavus(Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcusfuriosus (Pfu) DNA polymerase as well as Turbo Pfu DNA polymerase,Thermococcus litoralis (Tli) DNA polymerase or Vent DNA polymerase,Pyrococcus sp. GB-D polymerase, “Deep Vent” DNA polymerase, New EnglandBiolabs), Thermotoga maritima (Tma) DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD)DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNApolymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcusacidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase;Thermococcus sp. 9° N-7 DNA polymerase; Thermococcus sp. NA1;Pyrodictium occultum DNA polymerase; Methanococcus voltae DNApolymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;the heterodimeric DNA polymerase DP1/DP2, etc.

Suitable RNA polymerases include, without limitation, T3, T5, T7, andSP6 RNA polymerases.

Suitable reverse transcriptases include without limitation reversetranscriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV andMoMuLV, as well as the commercially available “Superscript” reversetranscriptases, (Life Technologies Corp., Carlsbad, Calif.) andtelomerases.

In some embodiments, the polymerase is selected from the groupconsisting of: Phi-29 DNA polymerase, a variant of Phi-29 DNApolymerase, B103 DNA polymerase and a variant of B103 DNA polymerase.

In another aspect, the polymerases can include one or more mutationsthat improve the performance of the polymerase in the particularbiological assay of interest. The mutations can include amino acidsubstitutions, insertions, or deletions.

Selecting a Polymerase

The selection of the polymerase for use in the disclosed methods can bebased on the desired polymerase behavior in the particular biologicalassay of interest. For example, the polymerase can be selected toexhibit enhanced or reduced activity in a particular assay, or enhancedor reduced interaction with one or more particular substrates.

For example, in some embodiments the polymerase is selected based on thepolymerization kinetics of the polymerase either in unconjugated form orwhen linked to a reporter moiety (labeled polymerase conjugate). Forexample, the polymerase can be a polymerase having altered nucleotidebinding and/or altered nucleotide incorporation kinetics which areselected on the basis of kinetic behavior relating to nucleotide binding(e.g., association), nucleotide dissociation (intact nucleotide),nucleotide fidelity, nucleotide incorporation (e.g., catalysis), and/orrelease of the cleavage product. The selected polymerase can bewild-type or mutant.

In one embodiment, polymerases may be selected that retain the abilityto selectively bind complementary nucleotides. In another embodiment,the polymerases may be selected which exhibit a modulated rate (fasteror slower) of nucleotide association or dissociation. In anotherembodiment, the polymerases may be selected which exhibit a reduced rateof nucleotide incorporation activity (e.g., catalysis) and/or a reducedrate of dissociation of the cleavage product and/or a reduced rate ofpolymerase translocation (after nucleotide incorporation). Some modifiedpolymerases which exhibit nucleotide binding and a reduced rate ofnucleotide incorporation have been described (Rank, U.S. publishedpatent application No. 2008/0108082; Hanzel, U.S. published patentapplication No. 2007/0196846).

In polymerases from different classes (including DNA-dependentpolymerases), an active-site lysine can interact with the phosphategroups of a nucleoside triphosphate molecule bound to the active site.The lysine residue has been shown to protonate the pyrophosphateleaving-group upon nucleotidyl transfer. Mutant polymerases having thislysine substituted with leucine, arginine, histidine or other aminoacids, exhibit greatly reduced nucleotide incorporation rates (Castro,et al., 2009 Nature Structural and Molecular Biology 16:212-218). Oneskilled in the art can use amino acid alignment and/or comparison ofcrystal structures of polymerases as a guide to determine which lysineresidue to replace with alternative amino acids. The sequences of Phi29(SEQ ID NOS: 6-12), RB69 (SEQ ID NO: 13), B103 (SEQ ID NOS: 1-5), andKlenow fragment can be used as the basis for selecting the amino acidresidues to be modified (for B103 polymerase, see Hendricks, et al.,U.S. Ser. No. 61/242,771, filed on Sep. 15, 2009, or U.S. Ser. No.61/293,618, filed on Jan. 8, 2010). In one embodiment, a modified phi29polymerase can include lysine at position 379 and/or 383 substitutedwith leucine, arginine or histidine.

In other embodiments, the polymerase can be selected based on thecombination of the polymerase and nucleotides, and the reactionconditions, to be used for the nucleotide binding and/or nucleotideincorporation reactions. For example, certain polymerases in combinationwith nucleotides which comprise 3, 4, 5, 6, 7, 8, 9, 10 or morephosphate groups can be selected for performing the disclosed methods.In another example, certain polymerases in combination with nucleotideswhich are linked to an energy transfer moiety can be selected forperforming the nucleotide incorporation methods.

The polymerases, nucleotides, and reaction conditions, can be screenedfor their suitability for use in the nucleotide binding and/ornucleotide incorporation methods, using well known screening techniques.For example, the suitable polymerase may be capable of bindingnucleotides and/or incorporating nucleotides. For example, the reactionkinetics for nucleotide binding, association, incorporation, and/ordissociation rates, can be determined using rapid kinetics techniques(e.g., stopped-flow or quench flow techniques). Using stopped-flow orquench flow techniques, the binding kinetics of a nucleotide can beestimated by calculating the 1/k_(d) value. Stopped-flow techniqueswhich analyze absorption and/or fluorescence spectroscopy properties ofthe nucleotide binding, incorporation, or dissociation rates to apolymerase are well known in the art (Kumar and Patel 1997 Biochemistry36:13954-13962; Tsai and Johnson 2006 Biochemistry 45:9675-9687; Hanzel,U.S. published patent application No. 2007/0196846). Other methodsinclude quench flow (Johnson 1986 Methods Enzymology 134:677-705),time-gated fluorescence decay time measurements (Korlach, U.S. Pat. No.7,485,424), plate-based assays (Clark, U.S. published patent applicationNo. 2009/0176233), and X-ray crystal structure analysis (Berman 2007EMBO Journal 26:3494). Nucleotide incorporation by a polymerase can alsobe analyzed by gel separation of the primer extension products. In oneembodiment, stopped-flow techniques can be used to screen and selectcombinations of nucleotides with polymerases having a t_(pol) value(e.g., 1/k_(pol)) which is less than a t⁻¹ (e.g., 1/k⁻¹) value.Stopped-flow techniques for measuring t_(pol) (MP Roettger 2008Biochemistry 47:9718-9727; M Bakhtina 2009 Biochemistry 48:3197-320) andt⁻¹ (M Bakhtina 2009 Biochemistry 48:3197-3208) are known in the art.

For example, some phi29 or B103 (SEQ ID NOS: 1, 2, or 3) polymerases(wild type or mutant) exhibit t_(pol) values which are less than t⁻1values, when reacted with tetraphosphate, pentaphosphate orhexaphosphate nucleotides. These polymerases can offer improvements indistinguishing between productive and non-productive nucleotide bindingevents compared to other polymerases. In another embodiment, polymerasescan be modified by binding it to a chemical compound or an antibody, inorder to inhibit nucleotide incorporation.

In some embodiments, the selection of the polymerase may be determinedby the level of processivity desired for conducting nucleotideincorporation or polymerization reactions. The polymerase processivitycan be gauged by the number of nucleotides incorporated for a singlebinding event between the polymerase and the target molecule base-pairedwith the polymerization initiation site. For example, the processivitylevel of the polymerase may be about 1, 5, 10, 20, 25, 50, 100, 250,500, 750, 1000, 2000, 5000, or 10,000 or more nucleotides incorporatedwith a single binding event. Processivity levels typically correlatewith read lengths of a polymerase. Optionally, the polymerase can beselected to retain the desired level of processivity when conjugated toa reporter moiety.

The selection of the polymerase may be determined by the level offidelity desired, such as the error rate per nucleotide incorporation.The fidelity of a polymerase may be partly determined by the 3′→5′exonuclease activity associated with a DNA polymerase. The fidelity of aDNA polymerase may be measured using assays well known in the art(Lundburg et al., 1991 Gene, 108:1-6). The error rate of the polymerasecan be one error per about 100, or about 250, or about 500, or about1000, or about 1500 incorporated nucleotides. In some embodiments, thepolymerase is selected to exhibit high fidelity. Such high-fidelitypolymerases include those exhibiting error rates typically of about5×10⁻⁶ per base pair or lower.

In some embodiments, the selection of the polymerase may be determinedby the rate of nucleotide incorporation such as about one nucleotide per2-5 seconds, or about one nucleotide per second, or about 5 nucleotidesper second, or about 10 nucleotides per second, or about 20 nucleotidesper second, or about 30 nucleotides per second, or more than 40nucleotides per second, or more than 50-100 per second, or more than 100per second. In one embodiment, polymerases exhibiting reduced nucleotideincorporation rates include mutant phi29 polymerase having lysinesubstituted with leucine, arginine, histidine or other amino acids(Castro 2009 Nature Structural and Molecular Biology 16:212-218).

In some embodiments, the polymerase can be selected to exhibit eitherreduced or enhanced rates of nucleotide incorporation when reacted withnucleotides linked at the terminal phosphate group with an energytransfer acceptor.

In some embodiments, the polymerase can be selected to exhibit eitherreduced or enhanced nucleotide binding times for a particular nucleotideof interest. In some embodiments, the nucleotide binding time of theselected polymerase for the particular labeled nucleotide of interestcan be between about 20 msec and about 300 msec, typically between about55 msec and about 100 msec. In some embodiments, the nucleotide bindingtime of the selected polymerase for the particular labeled nucleotide ofinterest can be between about 1.5 and about 4 times the nucleotidebinding time of the corresponding wild-type polymerase for the labelednucleotide. These polymerases can offer improvements in distinguishingbetween productive and non-productive nucleotide binding events comparedto other polymerases.

In some embodiments, the polymerase can be selected, mutated, modified,evolved or otherwise engineered to exhibit either reduced or enhancedentry of nucleotides, particularly labeled nucleotides, into thepolymerase active site. These polymerases can offer improvements indistinguishing between productive and non-productive nucleotide bindingevents compared to other polymerases.

In some embodiments, the polymerase can be selected to exhibit a reducedK_(sub) for a substrate, particularly a labeled nucleotide. In someembodiments, the polymerase can comprise one or more mutations resultingin altered K_(cat)/K_(sub) and/or V_(max)/K_(sub) for a particularlabeled nucleotide. In some embodiments, the K_(cat)/K_(sub), theV_(max)/K_(sub), or both, are increased compared to the wild typepolymerase.

In one embodiment, mutant polymerases having altered nucleotide bindingkinetics and/or altered nucleotide incorporation kinetics can beselected for use in the nucleotide incorporation methods. The alteredkinetics for nucleotide binding and/or for nucleotide incorporationinclude: polymerase binding to the target molecule; polymerase bindingto the nucleotide; polymerase catalyzing nucleotide incorporation; thepolymerase cleaving the phosphate group or substituted phosphate group;and/or the polymerase releasing the cleavage product. These polymerasescan offer improvements in distinguishing between productive andnon-productive nucleotide binding events compared to other polymerases.

In one embodiment, the selected polymerases can have improvedphoto-stability compared to polymerases traditionally used in nucleotidepolymerization reactions. The desirable polymerases can remainenzymatically active during and/or after exposure to electromagneticenergy (e.g., light). For example, the desirable polymerase can retain alevel of enzymatic activity, and/or be enzymatically active for agreater length of time, compared to polymerases traditionally used innucleotide polymerization reactions after exposure to electromagneticenergy. Methods for measuring enzymatic activity are well known in theart.

In one embodiment, the selected polymerase can be enzymatically activewhen conjugated to an energy transfer moiety (e.g., nanoparticle orfluorescent dye). The selected polymerase, as part of apolymerase-energy transfer moiety conjugate, can polymerize nucleotides.For example, various forms of B103 polymerase (SEQ ID NOS: 1, 2, and 3)retain enzymatic activity when linked to a nanoparticle or fluorescentdye. Conjugates having these types of selected polymerases offeradvantages over other polymerases which may lose most or all enzymaticactivity when linked to an energy transfer moiety.

In some embodiments, the polymerase can be a deletion mutant whichretains nucleotide polymerization activity but lacks the 3′→5′ or 5′→3′exonuclease activity (SEQ ID NOS: 1-12). For example, mutant phi29polymerases having exonuclease-minus activity, or reduced exonucleaseactivity, can optionally comprise the amino acid sequence of SEQ ID NOS:7-12 and further comprise one or more amino acid substitutions atpositions selected from the group consisting of: 12, 14, 15, 62, 66, 165and 169 (wherein the numbering is relative to the amino acid sequence ofwild type phi29 according to SEQ ID NO: 6). In some embodiments, thepolymerase is a phi29 polymerase comprising the amino acid sequence ofSEQ ID NO:6 and one or more of the following amino acid substitutions:D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C, and D169A, wherein thenumbering is relative to SEQ ID NO:6.

In one embodiment, the mutant phi29 polymerases include one or moreamino acid mutations at positions selected from the group consisting of:132, 135, 250, 266, 332, 342, 368, 371, 375, 379, 380, 383, 387, 390,458, 478, 480, 484, 486 and 512, wherein the numbering is relative tothe amino acid sequence of SEQ ID NO: 6. In some embodiments, the phi29polymerase can comprise an amino acid deletion, wherein the deletionincludes some of all of the amino acids spanning positions 306 to 311(relative to the numbering in SEQ ID NO: 6).

In one embodiment, the mutant phi29 polymerase includes one or moreamino acid mutations selected from the group consisting of: K132A,K135A, K135D, K135E, V250A, V250C, Y266F, D332Y, L342G, T368D, T368E,T368F, K370A, K371E, T372D, T372E, T372R, T372K, E375A, E375F, E375H,E375K, E375Q, E375R, E375S, E375W, E375Y, K379A, Q380A, K383E, K383H,K383L, K383R, N387Y, Y390F, D458N, K478D, K478E, K478R, L480K, L480R,A484E, E486A, E486D, K512A K512D, K512E, K512R, K512Y,K371E/K383E/N387Y/D458N, Y266F/Y390F, Y266F/Y390F/K379A/Q380A,K379A/Q380A, E375Y/Q380A/K383R, E375Y/Q380A/K383H, E375Y/Q380A/K383L,E375Y/Q380A/V250A, E375Y/Q380A/V250C, E375Y/K512Y/T368F,E375Y/K512Y/T368F/A484E, K379A/E375Y, K379A/K383R, K379A/K383H,K379A/K383L, K379A/Q380A, V250A/K379A, V250A/K379A/Q380A,V250C/K379A/Q380A, K132A/K379A and deletion of some or all of the aminoacid residues spanning R306 to K311, wherein the numbering is relativeto the amino acid sequence of SEQ ID NO: 6.

Without being bound to any particular theory, it is thought that thedomain comprising amino acid residues 304-314 of the amino acid sequenceof SEQ ID NO: 6 (Phi-29 polymerase), or homologs thereof, can reduce orotherwise interfere with DNA initiation and/or elongation by inhibitingaccess to the Phi-29 polymerase active site, and that this region mustbe displaced in order to allow access to the active site. See, e.g.,Kamtekar et al., “The Φ29 DNA polymerase:protein primer structuresuggests a model for the initiation to elongation transition”, EMBO J.,25:1335-1343 (2005).

In another embodiment, the polymerase can be a B103 polymerasecomprising the amino acid sequence of SEQ ID NOS: 1-5. The B103polymerase can optionally include one or more mutations that reduce theexonuclease activity of the polymerase. Optionally, such mutations caninclude any one or a combination of mutations at the following aminoacid positions: 2, 9, 11, 12, 14, 15, 58, 59, 63, 162, 166, 377 and 385,wherein the numbering is relative to SEQ ID NOS: 1 or 2. In someembodiments, the B103 polymerase can optionally comprise the amino acidsequence of SEQ ID NOS: 1 or 2, and further comprise one or more aminoacid substitutions selected from the group consisting of: D9A, E11A,E111, T121, H58R, N59D, D63A, Y162F, Y162C, D166A, Q377A and 5385G,wherein the numbering is relative to SEQ ID NOS: 1 or 2.

In some embodiments, the B103 polymerase can optionally the amino acidsequence of SEQ ID NOS: 1 or 2, and further comprise one or more aminoacid substitutions selected from the group consisting of (in singleletter amino acid code): H370G, H370T, H370S, H370K, H370R, H370A,H370Q, H370W, H370Y, H370F, E371G, E371H, E371T, E3715, E371K, E371R,E371A, E371Q, E371W, E371Y, E371F, K372G, K372E, K372T, K372S, K372R,K372A, K372Q, K372W, K372Y, K372F, K380E, K380T, K380S, K380R, K380A,K380Q, K380W, K380Y, K380F, D507H, D507G, D507E, D507T, D5075, D507R,D507A, D507R, D507Q, D507W, D507Y, D507F, K509H, K509G, K509D, K509R,K509E, K509T, K5095, K509R, K509A, K509Q, K509W, K509Y and K509F,wherein the numbering is relative to the sequence shown in SEQ ID NOS: 1or 2. The B103 polymerase can optionally further comprise the amino acidsequence of any of the polymerases disclosed by Hendricks, in U.S. Ser.No. 61/242,771, filed on Sep. 15, 2009, or U.S. Ser. No. 61/293,618,filed on Jan. 8, 2010.

Polymerases having desirable properties, including those having alterednucleotide binding and/or nucleotide incorporation kinetics, havingimproved photo-stability, and/or having improved enzymatic activity whenconjugated to an energy transfer moiety, include polymerases accordingto SEQ ID NOS: 1-5.

SEQ ID NO: 1MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 2MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 3MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTREKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVL VDSVFTIKSEQ ID NO: 4MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO:5 MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVL VDSVFTIKSEQ ID NO: 6MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 7MGLRRASLHHLLGGGGSGGGGSAAAGSAARKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 8MHHHHHHLLGGGGSGGGGSAAAGSAARKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNAIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 9MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO:10 MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKALAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO:11 MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNGLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO:12 MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVL VDDTFTIKSEQ ID NO: 13MKEFYLTVEQIGDSIFERYIDSNGRERTREVEYKPSLFAHCPESQATKYFDIYGKPCTRKLFANMRDASQWIKRMEDIGLEALGMDDFKLAYLSDTYNYEIKYDHTKIRVANFDIEVTSPDGFPEPSQAKHPIDAITHYDSIDDRFYVFDLLNSPYGNVEEWSIEIAAKLQEQGGDEVPSEIIDKIIYMPFDNEKELLMEYLNFWQQKTPVILTGWNVESFDIPYVYNRIKNIFGESTAKRLSPHRKTRVKVIENMYGSREIITLFGISVLDYIDLYKKFSFTNQPSYSLDYISEFELNVGKLKYDGPISKLRESNHQRYISYNIIDVYRVLQIDAKRQFINLSLDMGYYAKIQIQSVFSPIKTWDAIIFNSLKEQNKVIPQGRSHPVQPYPGAFVKEPIPNRYKYVMSFDLTSLYPSIIRQVNISPETIAGTFKVAPLHDYINAVAERPSDVYSCSPNGMMYYKDRDGVVPTEITKVFNQRKEHKGYMLAAQRNGEIIKEALHNPNLSVDEPLDVDYRFDFSDEIKEKIKKLSAKSLNEMLFRAQRTEVAGMTAQINRKLLINSLYGALGNVWFRYYDLRNATAITTFGQMALQWIERKVNEYLNEVCGTEGEAFVLYGDTDSIYVSADKIIDKVGESKFRDTNHWVDFLDKFARERMEPAIDRGFREMCEYMNNKQHLMFMDREAIAGPPLGSKGIGGFWTGKKRYALNVWDMEGTRYAEPKLKIMGLETQKSSTPKAVQKALKECIRRMLQEGEESLQEYFKEFEKEFRQLNYISIASVSSANNIAKYDVGGFPGPKCPFHIRGILTYNRAIKGNIDAPQVVEGEKVYVLPLREGNPFGDKCIAWPSGTEITDLIKDDVLHWMDYTVLLEKTFIKPLEGFTSAAKLDYEKKASLFDM FDFFusion Proteins

In one aspect, the polymerase can be a fusion protein comprising theamino acid sequence of a nucleic acid-dependent polymerase (thepolymerase portion) linked to the amino acid sequence of a second enzymeor a biologically active fragment thereof (the second enzyme portion).The second enzyme portion of the fusion protein may be linked to theamino or carboxyl end of the polymerase portion, or may be insertedwithin the polymerase portion. The polymerase portion of the fusionprotein may be linked to the amino or carboxyl end of the second enzymeportion, or may be inserted within the second enzyme portion. In someembodiments, the polymerase and second enzyme portions can be linked toeach other in a manner which does not significantly interfere withpolymerase activity of the fusion or with the ability of the fusion tobind nucleotides, or does not significantly interfere with the activityof the second enzyme portion. In the fusion protein, the polymeraseportion or the second enzyme portions can be linked with at least oneenergy transfer donor moiety. The fusion protein can be a recombinantprotein having a polymerase portion and a second enzyme portion. In someembodiments, the fusion protein can include a polymerase portionchemically linked to the second enzyme portion.

Evolved Polymerases

The polymerase can be a modified polymerase having certain desiredcharacteristics, such as an evolved polymerase selected from a directedor non-directed molecular evolution procedure. The evolved polymerasecan exhibit modulated characteristics or functions, such as changes in:affinity, specificity, or binding rates for substrates (e.g., targetmolecules, polymerization initiation sites, or nucleotides); bindingstability to the substrates (e.g., target molecules, polymerizationinitiation sites, or nucleotides); nucleotide incorporation rate;nucleotide permissiveness; exonuclease activity (e.g., 3′→5′ or 5′→3′);rate of extension; processivity; fidelity; stability; or sensitivityand/or requirement for temperature, chemicals (e.g., DTT), salts,metals, pH, or electromagnetic energy (e.g., excitation or emittedenergy). Many examples of evolved polymerases having altered functionsor activities can be found in U.S. provisional patent application No.61/020,995, filed Jan. 14, 2008.

Methods for creating and selecting proteins and enzymes having thedesired characteristics are known in the art, and include:oligonucleotide-directed mutagenesis in which a short sequence isreplaced with a mutagenized oligonucleotide; error-prone polymerasechain reaction in which low-fidelity polymerization conditions are usedto introduce point mutations randomly across a sequence up to about 1 kbin length (R. C. Caldwell, et al., 1992 PCR Methods and Applications2:28-33; H. Gramm, et al., 1992 Proc. Natl. Acad. Sci. USA89:3576-3580); and cassette mutagenesis in which a portion of a sequenceis replaced with a partially randomized sequence (A. R. Oliphant, etal., 1986 Gene 44:177-183; J. D. Hermes, et al., 1990 Proc. Natl. Acad.Sci. USA 87:696-700; A. Arkin and D. C. Youvan 1992 Proc. Natl. Acad.Sci. USA 89:7811-7815; E. R. Goldman and D. C. Youvan 1992Bio/Technology 10:1557-1561; Delagrave et al., 1993 Protein Engineering6: 327-331; Delagrave et al., 1993 Bio/Technology 11: 1548-155); anddomain shuffling.

Methods for creating evolved antibody and antibody-like polypeptides canbe adapted for creating evolved polymerases, and include appliedmolecular evolution formats in which an evolutionary design algorithm isapplied to achieve specific mutant characteristics. Many library formatscan be used for evolving polymerases including: phage libraries (J. K.Scott and G. P. Smith 1990 Science 249:386-390; S. E. Cwirla, et al.1990 Proc. Natl. Acad. Sci. USA 87:6378-6382; J. McCafferty, et al. 1990Nature 348:552-554) and lad (M. G. Cull, et al., 1992 Proc. Natl. Acad.Sci. USA 89:1865-1869).

Another adaptable method for evolving polymerases employs recombination(crossing-over) to create the mutagenized polypeptides, such asrecombination between two different plasmid libraries (Caren et al. 1994Bio/Technology 12: 517-520), or homologous recombination to create ahybrid gene sequence (Calogero, et al., 1992 FEMS Microbiology Lett. 97:41-44; Galizzi et al., WO91/01087). Another recombination methodutilizes host cells with defective mismatch repair enzymes (Radman etal., WO90/07576). Other methods for evolving polymerases include randomfragmentation, shuffling, and re-assembly to create mutagenizedpolypeptides (published application No. U.S. 2008/0261833, Stemmer).Adapting these mutagenesis procedures to generate evolved polymerases iswell within the skill of the art.

In some embodiments, the polymerase can be fused with, or otherwiseengineered to include, DNA-binding or other domains from other proteinsthat are capable of modulating DNA polymerase activity. For example,fusion of suitable portions of the Single-Stranded DNA Binding Protein(SSBP), thioredoxin and/or T7 DNA polymerase to bacterial or viral DNApolymerases has been shown to enhance both the processivity and fidelityof the DNA polymerase. Similarly, other groups have described efforts toengineer polymerases so as to broaden their substrate range. See, e.g.,Ghadessy et al, Nat. Biotech., 22 (6):755-759 (2004). Similarly, theconjugates of the present disclosure can optionally comprise anypolymerase engineered to provide suitable performance characteristics,including for example a polymerase fused to intact SSBP or fragmentsthereof, or to domains from other DNA-binding proteins (such as theherpes simplex virus UL42 protein.)

In some embodiments, a blend of different conjugates, each of whichcomprises a polymerase of unique sequence and characteristics, can beused according to the methods described herein. Use of such conjugateblends can additionally increase the fidelity and processivity of DNAsynthesis. For example, use of a blend of processive and non-processivepolymerases has been shown to result in increased overall read lengthduring DNA synthesis, as described in U.S. Published App. No.2004/0197800. Alternatively, conjugates comprising polymerases ofdifferent affinities for specific acceptor-labeled nucleotides can beused so as to achieve efficient incorporation of all four nucleotides.

In one embodiment, the polymerase can be a mutant which retainsnucleotide polymerization activity but lacks the 3′→5′ or 5′→3′exonuclease activity (SEQ ID NOS: 1-12). In another embodiment, thepolymerase can be an exonuclease minus mutant which is based on wildtype phi29 polymerase (SEQ ID NO: 6) (Blanco, U.S. Pat. Nos. 5,001,050,5,198,543, and 5,576,204; and Hardin PCT/US2009/31027 with anInternational filing date of Jan. 14, 2009) and comprising one or moresubstitution mutations, including: D12A, D66A, D169A, H61R, N62D, Q380A,and/or S388G, and any combination thereof.

In some embodiments, the polymerase can comprise the amino acid sequenceof any polymerase disclosed in U.S. Provisional Application No.61/242,771, filed on Sep. 15, 2009; 61/263,974, filed on Nov. 24, 2009and 61/299,919, filed on Jan. 29, 2010, or any variant thereof.

Polymerases Linked with Energy Transfer Moieties

The polymerase (or polymerase fusion protein) may be linked with atleast one energy transfer donor moiety. In the polymerase fusionprotein, the energy transfer donor moiety can be attached to thepolymerase portion or to the second enzyme portion. One or more energytransfer donor moieties can be linked to the polymerase (or polymerasefusion protein) at the amino end or carboxyl end or may be inserted inthe interior of the polymerase (or fusion protein sequence). The energytransfer donor moiety can be attached to the fusion protein in a mannerwhich does not interfere with the nucleotide binding activity, or withthe nucleotide incorporation activity, or with the activity of thesecond enzyme.

In one aspect, a single energy transfer donor moiety can be operablyattached with more than one polymerase (or more than one polymerasefusion protein) and the attachment can be at the amino end or carboxylend or may be inserted within the polymerase (or fusion proteinsequence).

In another aspect, a single energy transfer donor moiety can be linkedto one polymerase or polymerase fusion protein.

Target Nucleic Acid Molecules

The methods, compositions, systems and kits disclosed herein can involvethe use of target nucleic acid molecules. The target nucleic acidmolecule may be single or double-stranded molecules. The target nucleicacid molecules can be linear or circular. The target nucleic acidmolecules may be DNA, RNA or hybrid DNA-RNA molecules, DNA hairpins,DNA/RNA hybrids, or RNA hairpins. The target nucleic acid molecules maybe isolated in any form including chromosomal, genomic, organellar(e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules,cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA,oligonucleotide, or any type of nucleic acid library. The target nucleicacid molecules may be isolated from any source including from: organismssuch as prokaryotes, eukaryotes (e.g., humans, plants and animals),fungus, and viruses; cells; tissues; body fluids including blood, urine,serum, lymph, tumor, saliva, anal and vaginal secretions, amnioticsamples, perspiration, and semen; environmental samples; culturesamples; or synthesized nucleic acid molecules prepared usingrecombinant molecular biology or chemical synthesis methods.

The target nucleic acid molecules comprise naturally-occurringnucleotides, nucleotide variants, or any combination thereof. Forexample, the target molecules comprise alternate backbones, including:phosphoramidate; phosphorothioate; phosphorodithioate;O-methylphosphoroamidite linkages; and peptide nucleic acid backbonesand linkages. Other nucleic acids include those with bicyclic structuresincluding locked nucleic acids; positive backbones; non-ionic backbones;and non-ribose backbones.

The target nucleic acid molecules can carry a tag (e.g., His-tag), apolynucleotide tail (e.g., polynucleotide tail of A, G, C, T, or U), orcan be methylated. The target nucleic acid molecules may be nicked,sheared, or treated with an enzyme such as a restriction endonuclease ora nuclease. The target nucleic acid molecules can be about 10-50nucleotides, about 50-100 nucleotides, about 100-250 nucleotides, about250-500 nucleotides, or about 500-1000 nucleotides in length, or longer.The target nucleic acid molecules may be linked to an energy transfermoiety (e.g., donor or acceptor) or to a reporter moiety (e.g., dye)using methods well known in the art.

The target nucleic acid molecules can have a nucleotide sequence whichhas been previously determined or is unknown (e.g., de novo sequencing).The target molecule can be fragmented into shorter pieces and/ormodified for immobilization. Selection of the fragmentation andmodification technique may depend upon the desired fragment sizes andsubsequent preparation steps. Any combination of fragmentation and/ormodification techniques may be practiced in any order.

Single- or Double-Stranded Nucleic Acid Molecules

The target molecules can be single-stranded nucleic acid molecules whichare isolated by denaturing double-stranded molecules, or by chemicallysynthesizing single-stranded molecules. The target molecules can bedouble-stranded nucleic acid molecules. The single-stranded moleculescan be isolated away from double-stranded molecules by bead (e.g.,magnetic, biotinylated, or probe capture) attachment and enrichmentprocedures, CsCl gradient centrifugation methods, gel electrophoresis(e.g., polyacrylamide), or by capillary gel electrophoresis. The nucleicacid molecules can be attached to the beads via covalent or non-covalentlinkage.

Nucleic Acid Sample Preparation

The nucleic acid molecules, including the target molecules, primers, andoligonucleotides, may be isolated and modified at their ends and/or theinterior of the molecules using well known procedures, including:fragmentation, ligation, hybridization, enzymatic, and/or chemicalmodification, conjugation with an energy transfer (donor or acceptor) orreporter moiety, or any combination of these procedures.

Nucleic Acid Molecules—Fragmentation

Techniques which fragment the nucleic acid molecules at random orspecific sites, or a combination of these techniques can be used.

The nucleic acid molecules can be fragmented at random or specific sitesusing any fragmentation procedures. The nucleic acid molecules can befragmented using mechanical force, including: shear forces (e.g., smallorifice or a needle); nebulization (S. Surzycki 1990 In: “TheInternational Conference on the Status and Future of Research on theHuman Genome. Human Genome II”, San Diego, Calif., pp. 51; and S. J.Surzycki, 2000 in: “Basic Methods in Molecular Biology”, New York, N.Y.:Springer-Verlag); or sonication. For example, nucleic acid molecules canbe fragmented by sonicating in a COVARIS (e.g., Models S2, E210, orAFA).

The nucleic acid molecules can be chemically fragmented using, forexample: acid-catalyzed hydrolysis of the backbone and cleavage withpiperidine; internucleosomal DNA fragmentation using a copper (II)complex of 1,10-phenanthroline (o-phenanthroline, OP), CuII(OP)₂ in thepresence of ascorbic acid (Shui Ying Tsang 1996 Biochem. Journal317:13-16).

The nucleic acid molecules can be enzymatically fragmented using type I,II or II restriction endonucleases (N. E. Murray 2000 Microbiol. Mol.Biol. Rev. 64: 412-34; A. Pingoud and A. Jeltsch 2001 Nucleic Acids Res.29: 3705-27; D. T. Dryden, et al., 2001 Nucleic Acids Res. 29: 3728-41;and A. Meisel, et al., 1992 Nature 355: 467-9). Enzymatic cleavage ofDNA may include digestion using various ribo- and deoxyribonucleases orglycosylases. The nucleic acid molecules can be digested with DNase I orII. The nucleic acid fragments can be generated by enzymatically copyingan RNA template. Fragments can be generated using processive enzymaticdegradation (e.g., Si nuclease). The enzymatic reactions can beconducted in the presence or absence of salts (e.g., Mg²⁺, Mn²⁺, and/orCa²⁺), and the pH and temperature conditions can be varied according tothe desired rate of reaction and results, as is well known in the art.

Modified Nucleic Acid Molecules

The 5′ or 3′ overhang ends of a nucleic acid molecule can be convertedto blunt-ends using a “fill-in” procedure (e.g., dNTPS and DNApolymerase, Klenow, or Pfu or T4 polymerase) or using exonucleaseprocedure to digest away the protruding end.

The nucleic acid molecule ends can be ligated to one or moreoligonucleotides using DNA ligase or RNA ligase. The nucleic acidmolecules can be hybridized to one or more oligonucleotides. Theoligonucleotides can serve as linkers, adaptors, bridges, clamps,anchors, or capture oligonucleotides.

The oligonucleotides can be ligation-ready, having over-hang ends whichcan be ligated to the ends of the target molecules. The ligation-readyoligonucleotides can be used to circularize the target molecules.

A pair of oligonucleotides can include complementary sequences forhybridization. These paired oligonucleotides can be used as end-ligatedoligonucleotides to permit circularization of the target molecule. Thesepaired oligonucleotides can be used to hybridize to capture probesimmobilized on a surface.

The oligonucleotides can include sequences which are: enzyme recognitionsequences (e.g., restriction endonuclease recognition sites, DNA or RNApolymerase recognition sites); hybridization sites; or can include adetachable portion.

The oligonucleotide can be linked to a protein-binding molecule such asbiotin or streptavidin.

The oligonucleotides can be 4-20 nt/bp in length, or 20-40 nt/bp inlength, or 40-60 nt/bp in length, or longer.

Enzymatic and Chemical Modifications

The nucleic acid molecules can be methylated, for example, to conferresistance to restriction enzyme digestion (e.g., EcoRI).

The nucleic acid molecule ends can be phosphorylated ordephosphorylated.

A nick can be introduced into the nucleic acid molecules using, forexample DNase I. A pre-designed nick site can be introduced in dsDNAusing a double stranded probe, type II restriction enzyme, ligase, anddephosphorylation (Fu Dong-Jing, 1997 Nucleic Acids Research25:677-679).

A nick can be repaired using polymerase (e.g., DNA pol I or phi29),ligase (e.g., T4 ligase) and kinase (polynucleotide kinase).

A poly tail can be added to the 3′ end of the fragment using terminaltransferase (e.g., polyA, polyG, polyC, polyT, or polyU).

The target nucleic acid molecule can include pre-existing methylationsites. The target molecule can be modified using bisulfite treatment(e.g., disodium bisulfite) to convert unmethylated cytosines to uracils,which permits detection of methylated cytosines using, for example,methylation specific procedures (e.g., PCR or bisulfite genomicsequencing).

Size Selection

The nucleic acid molecules can be size selected, or the desired nucleicacid molecules can separated from undesirable molecules, using any artknown methods, including gel electrophoresis, size exclusionchromatography (e.g., spin columns), sucrose sedimentation, or gradientcentrifugation. Very large nucleic acid molecules, including wholechromosomes, can be size separated using pulsed-field gelelectrophoresis (Schwartz and Cantor 1984 Cell, 37: 67-75).

Amplification

The nucleic acid molecules can be amplified using methods, including:polymerase chain reaction (PCR); ligation chain reaction, which issometimes referred to as oligonucleotide ligase amplification (OLA);cycling probe technology (CPT); strand displacement assay (SDA);transcription mediated amplification (TMA); nucleic acid sequence basedamplification (NASBA); rolling circle amplification (RCA); and invasivecleavage technology.

Enrichment

Undesired compounds, or undesired fragments, can be removed or separatedfrom the desired target nucleic acid molecules to facilitate enrichmentof the desired target molecules. Enrichment methods can be achievedusing well known methods, including gel electrophoresis, chromatography,or solid phase immobilization (reversible or non-reversible). Forexample, AMPURE beads (Agencourt) can bind DNA fragments but not bindunincorporated nucleotides, free primers, DNA polymerases, and salts,thereby facilitating enrichment of the desired DNA fragments.

Embodiments of the Target Molecule

In one embodiment, the target molecule can be a recombinant DNA moleculewhich is a self-priming hairpin oligonucleotide. The hairpinoligonucleotide can be linked at the 5′ or 3′ end, or internally, to atleast one molecule of a binding partner (e.g., biotin). The biotinmolecule can be used to immobilize the hairpin oligonucleotide to thesurface (via avidin-like molecule), or for attachment to a reportermoiety. The hairpin oligonucleotide can be linked to at least one energytransfer moiety, such as a fluorescent dye or a nanoparticle.

In another embodiment, a plurality of target nucleic acid molecules canbe linked to a solid surface (via the 5′ or 3′ end, or via an internalsite) to form a DNA curtain (see Greene, U.S. published patentapplication No. 2008/0274905, published on Nov. 6, 2008; and Fazio, etal., 2008 Langmuir 24:10524-10531). The nucleotide incorporation methodscan be practiced on the DNA curtain in an aqueous flowing condition.

Reporter Moieties

The methods, systems, compositions and kits disclosed herein can involvethe use of one or more reporter moieties which are linked to the solidsurfaces, nanoparticles, polymerases, nucleotides, target nucleic acidmolecules, primers, and/or oligonucleotides.

The reporter moieties may be selected so that each absorbs excitationradiation and/or emits fluorescence at a wavelength distinguishable fromthe other reporter moieties to permit monitoring the presence ofdifferent reporter moieties in the same reaction. Two or more differentreporter moieties can be selected having spectrally distinct emissionprofiles, or having minimal overlapping spectral emission profiles.

In one aspect, the signals (e.g., energy transfer signals) from thedifferent reporter moieties do not significantly overlap or interfere,by quenching, colorimetric interference, or spectral interference.

The chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate,3-indoxyl phosphate, p-nitrophenyl phosphate, β-lactamase,peroxidase-based chemistry, and derivatives thereof.

The chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetanecompound. The 1,2-dioxetane compound includes disodium2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chloro-)tricyclo[3,3,1-1^(3,7)]-decan]-1-yl)-1-phenyl phosphate (e.g.,CDP-STAR), chloroadamant-2′-ylidenemethoxyphenoxy phosphorylateddioxetane (e.g., CSPD), and3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane(e.g., AMPPD).

In some embodiments, the fluorescent moiety can optionally include:rhodols; resorufins;

-   -   coumarins; xanthenes; acridines; fluoresceins; rhodamines;        erythrins; cyanins; phthalaldehydes; naphthylamines;        fluorescamines; benzoxadiazoles; stilbenes; pyrenes; indoles;        borapolyazaindacenes; quinazolinones; eosin; erythrosin;        Malachite green; CY dyes (GE Biosciences), including Cy3 (and        its derivatives) and Cy5 (and its derivatives); DYOMICS and        DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632,        DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680,        DY-682, DY-701, DY-734, DY-752, DY-777 and DY-782; Lucifer        Yellow; CASCADE BLUE; TEXAS RED; BODIPY (boron-dipyrromethene)        (Molecular Probes) dyes including BODIPY 630/650 and BODIPY        650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO        465, ATTO 610 611X, ATTO 610 (N-succinimidyl ester), ATTO 635        (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR        647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and        ALEXA FLUOR 680 (Molecular Probes); DDAO        (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any        derivatives thereof) (Molecular Probes); QUASAR dyes        (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS        ester), IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester);        EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied        Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes        (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS        RED (Molecular Devices); SUNNYVALE RED (Molecular Devices);        LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including        EPOCH REDMOND RED (phosphoramidate), EPOCH YAKIMA YELLOW        (phosphoramidate), EPOCH GIG HARBOR GREEN (phosphoramidate);        Tokyo green (M. Kamiya, et al., 2005 Angew. Chem. Int. Ed.        44:5439-5441); and CF dyes including CF 647 and CF555 (Biotium).

Quencher dyes may include: ATTO 540Q, ATTO 580Q, and ATTO 612Q(Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35(Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (GlenResearch). The fluorescent moiety can be a 7-hydroxycoumarin-hemicyaninehybrid molecule which is a far-red emitting dye (Richard 2008 Org. Lett.10:4175-4178).

The fluorescent moiety may be a fluorescence-emitting metal such as alanthanide complex, including those of Europium and Terbium.

A number of examples of fluorescent moieties are found in PCTpublication WO/2008/030115, and in Haugland, Molecular Probes Handbook,(Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.),Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum PressNew York (1999).

In one aspect, the reporter moieties can be energy transfer moieties.

FRET

In some embodiments, the methods, compositions, systems and kitsdisclosed herein can involve the use of one or more moieties capable ofundergoing energy transfer. Such energy transfer moieties can includeenergy transfer donors and acceptors. The energy transfer moieties canbe linked to the solid surfaces, nanoparticles, polymerases,nucleotides, target nucleic acid molecules, primers, and/oroligonucleotides.

In one aspect, the energy transfer moiety can be an energy transferdonor. For example, the energy transfer donor can be a nanoparticle oran energy transfer donor moiety (e.g., fluorescent dye). In anotheraspect, the energy transfer moiety can be an energy transfer acceptor.For example, the energy transfer acceptor can be an energy acceptor dye.In another aspect, the energy transfer moiety can be a quencher moiety.

In one aspect, the energy transfer pair can be linked to the samemolecule. For example, the energy transfer donor and acceptor pair canbe linked to a single polymerase, which can provide detection ofconformational changes in the polymerase. In another aspect, the donorand acceptor can be linked to different molecules in any combination.For example, the donor can be linked to the polymerase, target molecule,or primer molecule, and/or the acceptor can be linked to the nucleotide,the target molecule, or the primer molecule.

The energy transfer donor is capable of absorbing electromagnetic energy(e.g., light) at a first wavelength and emitting excitation energy inresponse. The energy acceptor is capable of absorbing excitation energyemitted by the donor and fluorescing at a second wavelength in response.

The donor and acceptor moieties can interact with each other physicallyor optically in a manner which produces a detectable signal (e.g.,energy transfer signal) when the two moieties are in proximity with eachother. A proximity event includes two different moieties (e.g., energytransfer donor and acceptor) approaching each other, or associating witheach other, or binding each other.

The donor and acceptor moieties can transfer energy in various modes,including: fluorescence resonance energy transfer (FRET) (L. Stryer 1978Ann. Rev. Biochem. 47: 819-846; Schneider, U.S. Pat. No. 6,982,146;Hardin, U.S. Pat. No. 7,329,492; Hanzel U.S. published patentapplication No. 2007/0196846), scintillation proximity assays (SPA)(Hart and Greenwald 1979 Molecular Immunology 16:265-267; U.S. Pat. No.4,658,649), luminescence resonance energy transfer (LRET) (G. Mathis1995 Clin. Chem. 41:1391-1397), direct quenching (Tyagi et al, 1998Nature Biotechnology 16:49-53), chemiluminescence energy transfer (CRET)(Campbell and Patel 1983 Biochem. Journal 216:185-194), bioluminescenceresonance energy transfer (BRET) (Y. Xu, et al., 1999 Proc. Natl. Acad.Sci. 96:151-156), and excimer formation (J. R. Lakowicz 1999 “Principlesof Fluorescence Spectroscopy”, Kluwer Academic/Plenum Press, New York).

In one exemplary embodiment, the energy transfer moieties can be a FRETdonor/acceptor pair. FRET is a distance-dependent radiationlesstransmission of excitation energy from a first moiety, referred to as adonor moiety, to a second moiety, referred to as an acceptor moiety.Typically, the efficiency of FRET energy transmission is dependent onthe inverse sixth-power of the separation distance between the donor andacceptor, r. For a typical donor-acceptor pair, r can vary betweenapproximately 10-100 Angstroms. FRET is useful for investigating changesin proximity between and/or within biological molecules. In someembodiments, FRET efficiency may depend on donor-acceptor distance r as1/r⁶ or 1/r⁴. The efficiency of FRET energy transfer can sometimes bedependent on energy transfer from a point to a plane which varies by thefourth power of distance separation (E. Jares-Erijman, et al., 2003 Nat.Biotechnol. 21:1387). The distance where FRET efficiency is 50% istermed R₀, also know as the Forster distance. R₀ is unique for eachdonor-acceptor combination and may be about 1-20 nm, or about 1-10 nm,or about 1-5 nm, or about 5-10 nm. A change in fluorescence from a donoror acceptor during a FRET event (e.g., increase or decrease in thesignal) can be an indication of proximity between the donor andacceptor.

In biological applications, FRET can provide an on-off type signalindicating when the donor and acceptor moieties are proximal (e.g.,within R₀) of each other. Additional factors affecting FRET efficiencyinclude the quantum yield of the donor, the extinction coefficient ofthe acceptor, and the degree of spectral overlap between the donor andacceptor. Procedures are well known for maximizing the FRET signal anddetection by selecting high yielding donors and high absorbing acceptorswith the greatest possible spectral overlap between the two (D. W.Piston and G. J. Kremers 2007 Trends Biochem. Sci. 32:407). Resonanceenergy transfer may be either an intermolecular or intramolecular event.Thus, the spectral properties of the energy transfer pair as a whole,change in some measurable way if the distance and/or orientation betweenthe moieties are altered.

The production of signals from FRET donors and acceptors can besensitive to the distance between donor and acceptor moieties, theorientation of the donor and acceptor moieties, and/or a change in theenvironment of one of the moieties (Deuschle et al. 2005 Protein Science14: 2304-2314; Smith et al. 2005 Protein Science 14:64-73). For example,a nucleotide linked with a FRET moiety (e.g., acceptor) may produce adetectable signal when it approaches, associates with, or binds apolymerase linked to a FRET moiety (e.g., donor). In another example, aFRET donor and acceptor linked to one protein can emit a FRET signalupon conformational change of the protein. Some FRET donor/acceptorpairs exhibit changes in absorbance or emission in response to changesin their environment, such as changes in pH, ionic strength, ionic type(NO₂. Ca⁺², Mg⁺², Zn⁺², Na⁺, Cl⁻, K⁺), oxygen saturation, and solvationpolarity.

The FRET donor and/or acceptor may be a fluorophore, luminophore,chemiluminophore, bioluminophore, or quencher (P. Selvin 1995 MethodsEnzymol 246:300-334; C. G. dos Remedios 1995 J. Struct. Biol.115:175-185; P. Wu and L. Brand 1994 Anal Biochem 218:1-13).

In some embodiments, the energy transfer moieties may not undergo FRET,but may undergo other types of energy transfer with each other,including luminescence resonance energy transfer, bioluminescenceresonance energy transfer, chemiluminescence resonance energy transfer,and similar types of energy transfer not strictly following theForster's theory, such as the non-overlapping energy transfer whennon-overlapping acceptors are utilized (Laitala and Hemmila 2005 Anal.Chem. 77: 1483-1487).

In one embodiment, the polymerase can be linked to an energy transferdonor moiety. In another embodiment, the nucleotide can be linked to anenergy transfer acceptor moiety. For example, in one embodiment thenucleotide comprises a polyphosphate chain and an energy transfer moietylinked to the terminal phosphate group of the polyphosphate chain. Achange in a fluorescent signal can occur when the labeled nucleotide isproximal to the labeled polymerase.

In one embodiment, when an acceptor-labeled nucleotide is proximal to adonor-labeled polymerase, the signal emitted by the donor moietydecreases. In another embodiment, when the acceptor-labeled nucleotideis proximal to the donor-labeled polymerase, the signal emitted by theacceptor moiety increases. In another embodiment, a decrease in donorsignal and increase in acceptor signal correlates with nucleotidebinding to the polymerase and/or correlates with polymerase-dependentnucleotide incorporation.

Quenchers

The energy transfer moiety can be a FRET quencher. Typically, quenchershave an absorption spectrum with large extinction coefficients, howeverthe quantum yield for quenchers is reduced, such that the quencher emitslittle to no light upon excitation. Quenching can be used to reduce thebackground fluorescence, thereby enhancing the signal-to-noise ratio. Inone aspect, energy transferred from the donor may be absorbed by thequencher which emits moderated (e.g., reduced) fluorescence. In anotheraspect, the acceptor can be a non-fluorescent chromophore which absorbsthe energy transferred from the donor and emits heat (e.g., the energyacceptor is a dark quencher).

For an example, a quencher can be used as an energy acceptor with ananoparticle donor in a FRET system, see I. L. Medintz, et al., 2003Nature Materials 2:630. One exemplary method involves the use ofquenchers in conjunction with reporters comprising fluorescent reportermoieties. In this strategy, certain nucleotides in the reaction mixtureare labeled with a reporter comprising a fluorescent label, while theremaining nucleotides are labeled with one or more quenchers.Alternatively, each of the nucleotides in the reaction mixture islabeled with one or more quenchers. Discrimination of the nucleotidebases is based on the wavelength and/or intensity of light emitted fromthe FRET acceptor, as well as the intensity of light emitted from theFRET donor. If no signal is detected from the FRET acceptor, acorresponding reduction in light emission from the FRET donor indicatesincorporation of a nucleotide labeled with a quencher. The degree ofintensity reduction may be used to distinguish between differentquenchers.

Examples of fluorescent donors and non-fluorescent acceptor (e.g.,quencher) combinations have been developed for detection of proteolysis(Matayoshi 1990 Science 247:954-958) and nucleic acid hybridization (L.Morrison, in: Nonisotopic DNA Probe Techniques, ed., L. Kricka, AcademicPress, San Diego, (1992) pp. 31 1-352; S. Tyagi 1998 Nat. Biotechnol.16:49-53; S. Tyagi 1996 Nat. Biotechnol. 14:947-8). FRET donors,acceptors and quenchers can be moieties which absorb electromagneticenergy (e.g., light) at about 300-900 nm, or about 350-800 nm, or about390-800 nm.

Materials for Energy Transfer Moieties

Energy transfer donor and acceptor moieties can be made from materialswhich typically fall into four general categories (see the review in: K.E. Sapford, et al., 2006 Angew. Chem. Int. Ed. 45:4562-4588), including:(1) organic fluorescent dyes, dark quenchers and polymers (e.g.,dendrimers); (2) inorganic material such as metals, metal chelates andsemiconductors nanoparticles; (3) biomolecules such as proteins andamino acids (e.g., green fluorescent protein and derivatives thereof);and (4) enzymatically catalyzed bioluminescent molecules. The materialfor making the energy transfer donor and acceptor moieties can beselected from the same or different categories.

The FRET donor and acceptor moieties which are organic fluorescent dyes,quenchers or polymers can include traditional dyes which emit in the UV,visible, or near-infrared region. The UV emitting dyes includecoumarin-, pyrene-, and naphthalene-related compounds. The visible andnear-infrared dyes include xanthene-, fluorescein-, rhodol-, rhodamine-,and cyanine-related compounds. The fluorescent dyes also includes DDAO((7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)), resorufin,ALEXA FLUOR and BODIPY dyes (both Molecular Probes), HILYTE Fluors(AnaSpec), ATTO dyes (Atto-Tec), DY dyes (Dyomics GmbH), TAMRA (PerkinElmer), tetramethylrhodamine (TMR), TEXAS RED, DYLIGHT (Thermo FisherScientific), FAM (AnaSpec), JOE and ROX (both Applied Biosystems), andTokyo Green.

Additional fluorescent dyes which can be used as quenchers includes:DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ (BiosearchTechnologies), QXL (AnaSpec), BBQ (Berry and Associates) and CYSQ/7Q(Amersham Biosciences).

The FRET donor and acceptor moieties which comprise inorganic materialsinclude gold (e.g., quencher), silver, copper, silicon, semiconductornanoparticles, and fluorescence-emitting metal such as a lanthanidecomplex, including those of Europium and Terbium.

Suitable FRET donor/acceptor pairs include: FAM as the donor and JOE,TAMRA, and ROX as the acceptor dyes. Other suitable pairs include: CYAas the donor and R6G, TAMRA, and ROX as the donor dyes. Other suitabledonor/acceptor pairs include: a nanoparticle as the donor, and ALEXAFLUORS dyes (e.g., 610, 647, 660, 680, 700). DYOMICS dyes, such as 634and 734 can be used as energy transfer acceptor dyes.

Nanoparticles

The methods, compositions, systems and kits disclosed herein can involvethe use of any suitable nanoparticles which can serve as donorfluorophores in energy transfer reactions such as FRET.

The nanoparticles can be attached to the solid surface or to anycomponent of the nucleotide incorporation or nucleotide polymerizationreactions in any combination (e.g., polymerases, nucleotides, targetnucleic acid molecules, primers, and/or oligonucleotides).

“Nanoparticle” may refer to any particle with at least one majordimension in the nanosize range. In general, nanoparticles can be madefrom any suitable metal (e.g., noble metals, semiconductors, etc.)and/or non-metal atoms. Nanoparticles can have different shapes, each ofwhich can have distinctive properties including spatial distribution ofthe surface charge; orientation dependence of polarization of theincident light wave; and spatial extent of the electric field. Theshapes include, but are not limited to: spheres, rods, discs, triangles,nanorings, nanoshells, tetrapods, nanowires, etc.

In one embodiment, the nanoparticle can be a core/shell nanoparticlewhich typically comprises a core nanoparticle surrounded by at least oneshell. For example, the core/shell nanoparticle can be surrounded by aninner and outer shell. In another embodiment, the nanoparticle is a corenanoparticle which has a core but no surrounding shell. The outmostsurface of the core or shell can be coated with tightly associatedligands which are not removed by ordinary solvation.

Examples of a nanoparticle include a nanocrystal, such as a core/shellnanocrystal, plus any associated organic ligands (which are not removedby ordinary solvation) or other materials which may coat the surface ofthe nanocrystal. In one embodiment, a nanoparticle has at least onemajor dimension ranging from about 1 to about 1000 nm. In otherembodiments, a nanoparticle has at least one major dimension rangingfrom about 1 to about 20 nm, about 1 to about 15 nm, about 1 to about 10nm or about 1 to 5 nm.

In some embodiments, a nanoparticle can have a layer of ligands on itssurface which can further be cross-linked to each other. In someembodiments, a nanoparticle can have other or additional surfacecoatings which can modify the properties of the particle, for example,increasing or decreasing solubility in water or other solvents. Suchlayers on the surface are included in the term ‘nanoparticle.’

In one embodiment, nanoparticle can refer to a nanocrystal having acrystalline core, or to a core/shell nanocrystal, and may be about 1 nmto about 100 nm in its largest dimension, about 1 nm to about 20 nm,about 1 nm to about 15 nm, about 1 nm to about 10 nm or preferably about5 nm to about 10 nm in its largest dimension. Small nanoparticles aretypically less than about 20 nm in their largest dimension.

“Nanocrystal” as used herein can refer to a nanoparticle made out of aninorganic substance that typically has an ordered crystalline structure.It can refer to a nanocrystal having a crystalline core (corenanocrystal) or to a core/shell nanocrystal.

A core nanocrystal is a nanocrystal to which no shell has been applied.Typically, it is a semiconductor nanocrystal that includes a singlesemiconductor material. It can have a homogeneous composition or itscomposition can vary with depth inside the nanocrystal.

A core/shell nanocrystal is a nanocrystal that includes a corenanocrystal and a shell disposed over the core nanocrystal. Typically,the shell is a semiconductor shell that includes a single semiconductormaterial. In some embodiments, the core and the shell of a core/shellnanocrystal are composed of different semiconductor materials, meaningthat at least one atom type of a binary semiconductor material of thecore of a core/shell is different from the atom types in the shell ofthe core/shell nanocrystal.

The semiconductor nanocrystal core can be composed of a semiconductormaterial (including binary, ternary and quaternary mixtures thereof),from: Groups II-VI of the periodic table, including ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V, including GaN,GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS; and/orGroup IV, including Ge, Si, Pb.

The semiconductor nanocrystal shell can be composed of materials(including binary, ternary and quaternary mixtures thereof) comprising:ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP,GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP,or AlSb.

Many types of nanocrystals are known, and any suitable method for makinga nanocrystal core and applying a shell to the core may be employed.Nanocrystals can have a surface layer of ligands to protect thenanocrystal from degradation in use or during storage.

“Quantum dot” as used herein refers to a crystalline nanoparticle madefrom a material which in the bulk is a semiconductor or insulatingmaterial, which has a tunable photophysical property in the nearultraviolet (UV) to far infrared (IR) range.

“Water-soluble” or “water-dispersible” is used herein to mean the itemcan be soluble or suspendable in an aqueous-based solution, such as inwater or water-based solutions or buffer solutions, including those usedin biological or molecular detection systems as known by those skilledin the art. While water-soluble nanoparticles are not truly ‘dissolved’in the sense that term is used to describe individually solvated smallmolecules, they are solvated (via hydrogen, electrostatic or othersuitable physical/chemical bonding) and suspended in solvents which arecompatible with their outer surface layer, thus a nanoparticle which isreadily dispersed in water is considered water-soluble orwater-dispersible. A water-soluble nanoparticle can also be consideredhydrophilic, since its surface is compatible with water and with watersolubility.

“Hydrophobic nanoparticle” as used herein refers to a nanoparticle whichis readily dispersed in or dissolved in a water-immiscible solvent likehexanes, toluene, and the like. Such nanoparticles are generally notreadily dispersed in water.

“Hydrophilic” as used herein refers to a surface property of a solid, ora bulk property of a liquid, where the solid or liquid exhibits greatermiscibility or solubility in a high-dielectric medium than it does in alower dielectric medium. By way of example, a material which is moresoluble in methanol than in a hydrocarbon solvent such as decane wouldbe considered hydrophilic.

“Coordinating solvents” as used herein refers to a solvent such as TDPA,OP, TOP, TOPO, carboxylic acids, and amines, which are effective tocoordinate to the surface of a nanocrystal. ‘Coordinating solvents’ alsoinclude phosphines, phosphine oxides, phosphonic acids, phosphinicacids, amines, and carboxylic acids, which are often used in growthmedia for nanocrystals, and which form a coating or layer on thenanocrystal surface. Coordinating solvents can exclude hydrocarbonsolvents such as hexanes, toluene, hexadecane, octadecene and the like,which do not have heteroatoms that provide bonding pairs of electrons tocoordinate with the nanocrystal surface. Hydrocarbon solvents which donot contain heteroatoms such as O, S, N or P to coordinate to ananocrystal surface are referred to herein as non-coordinating solvents.Note that the term ‘solvent’ is used in its ordinary way in these terms:it refers to a medium which supports, dissolves or disperses materialsand reactions between them, but which does not ordinarily participate inor become modified by the reactions of the reactant materials. However,in certain instances, the solvent can be modified by the reactionconditions. For example, TOP may be oxidized to TOPO, or a carboxylicacid can be reduced to an alcohol.

As used herein, the term “population” refers to a plurality ofnanoparticles having similar physical and/or optical properties.‘Population’ can refer to a solution or structure with more than onenanoparticle at a concentration suitable for single molecule analysis.In some embodiments, the population can be monodisperse and can exhibitless than at least 15% rms deviation in diameter of the nanoparticles,and spectral emissions in a narrow range of no greater than about 75 nmfull width at half max (FWHM). In the context of a solution, suspension,gel, plastic, or colloidal dispersion of nanoparticles, the nature ofthe population can be further characterized by the number ofnanoparticles present, on average, within a particular volume of theliquid or solid, or the concentration. In a two-dimensional format suchas an array of nanoparticles adhered to a solid substrate, the conceptof concentration is less convenient than the related measure of particledensity, or the number of individual particles per two-dimensional area.In this case, the maximum density would typically be that obtained bypacking particles “shoulder-to-shoulder” in an array. The actual numberof particles in this case would vary due to the size of the particles—agiven array could contain a large number of small particles or a smallnumber of larger particles.

As used herein, the terms “moderate to high excitation” refers tomonochromatic illumination or excitation (e.g., laser illumination)having a high power intensity sufficiently high such that the absorbedphotons per second for a given sample is between about 200,000 and about1,600,000.

In one aspect, the nanoparticle is a semiconductor nanoparticle havingsize-dependent optical and electronic properties. For example, thenanoparticle can emit a fluorescent signal in response to excitationenergy. The spectral emission of the nanoparticle can be tunable to adesired energy by selecting the particle size, size distribution, and/orcomposition of the semiconductor nanoparticle. For example, depending onthe dimensions, the semiconductor nanoparticle can be a fluorescentnanoparticle which emits light in the UV-visible-IR spectrum. The shellmaterial can have a bandgap greater than the bandgap of the corematerial.

In one aspect, the nanoparticle is an energy transfer donor. Thenanoparticle can be excited by an electromagnetic source such as a laserbeam, multi-photon excitation, or electrical excitation. The excitationwavelength can range between about 190 to about 800 nm including allvalues and ranges there in between. In some embodiments, thenanoparticle can be excited by an energy source having a wavelength ofabout 405 nm. In other embodiments, in response to excitation, thenanoparticle can emit a fluorescent signal at about 400-800 nm, or about605 nm.

In one aspect, the nanoparticle can undergo Raman scattering whensubjected to an electromagnetic source (incident photon source) such asa laser beam. The scattered photons have a frequency that is differentfrom the frequency of the incident photons. As result, the wavelength ofthe scattered photons is different than the incident photon source. Inone embodiment, the nanoparticle can be attached to a suitable tag orlabel to enhance the detectability of the nanoparticle via Ramanspectroscopy. The associated tag can be fluorescent or nonfluorescent.Such approaches can be advantageous in avoiding problems that can arisein the context of fluorescent nanoparticles, such as photobleaching andblinking. See, e.g., Sun et al., “Surface-Enhanced Raman ScatteringBased Nonfluorescent Probe for Multiplex DNA Detection”, Anal. Chem.79(11):3981-3988 (2007)

In one aspect, the nanoparticle is comprised of a multi-shell layeredcore which is achieved by a sequential shell material depositionprocess, where one shell material is added at a time, to provide ananoparticle having a substantially uniform shell of desired thicknesswhich is substantially free of defects. The nanoparticle can be preparedby sequential, controlled addition of materials to build and/or applyinglayers of shell material to the core. See e.g., U.S. PCT ApplicationSerial No. PCT/US09/061951 which is incorporated herein by reference asif set forth in full.

In another aspect, a method is provided for making a nanoparticlecomprising a core and a layered shell, where the shell comprises atleast one inner shell layer and at least one outer shell layer. Themethod comprises the steps: (a) providing a mixture comprising a core,at least one coordinating solvent; (b) heating the mixture to atemperature suitable for formation of an inner shell layer; (c) adding afirst inner shell precursor alternately with a second inner shellprecursor in layer additions, to form an inner shell layer which is adesired number of layers thick; (d) heating the mixture to a temperaturesuitable for formation of an outer shell layer; and (e) adding a firstouter shell precursor alternately with a second outer shell precursor inlayer additions, to form an outer shell layer which is a desired numberof layers thick. In one embodiment, if the coordinating solvent of (a)is not amine, the method further comprises an amine in (a).

In one aspect, at least one coordinating solvent comprises atrialkylphosphine, a trialkylphosphine oxide, phosphonic acid, or amixture of these. In another aspect, at least one coordinating solventcomprises trioctylphosphine (TOP), trioctylphosphine oxide (TOPO),tetradecylphosphonic acid (TDPA), or a mixture of these. In yet anotheraspect, the coordinating solvent comprises a primary or secondary amine,for example, decylamine, hexadecylamine, or dioctylamine.

In one aspect, the nanoparticle comprises a core comprising CdSe. Inanother aspect, the nanoparticle shell can comprise YZ wherein Y is Cdor Zn, and Z is S, or Se. In one embodiment, at least one inner shelllayer comprises CdS, and the at least one outer shell layer comprisesZnS.

In one aspect, the first inner shell precursor is Cd(OAc)₂ and thesecond inner shell precursor is bis(trimethylsilyl)sulfide (TMS2S). Inother aspects, the first and second inner shell precursors are added asa solution in trioctylphosphine (TOP). In other aspects, the first outershell precursor is diethylzinc (Et2Zn) and the second inner shellprecursor is dimethyl zinc (TMS2S). Sometimes, the first and secondouter shell precursors are added as a solution in trioctylphosphine(TOP).

In one aspect, the nanoparticle can have ligands which coat the surface.The ligand coating can comprise any suitable compound(s) which providesurface functionality (e.g., changing physicochemical properties,permitting binding and/or other interaction with a biomolecule, etc.).In some embodiments, the disclosed nanoparticle has a surface ligandcoating (in direct contact with the external shell layer) that addsvarious functionalities which facilitate it being water-dispersible orsoluble in aqueous solutions. There are a number of suitable surfacecoatings which can be employed to permit aqueous dispersibility of thedescribed nanoparticle. For example, the nanoparticle(s) disclosedherein can comprise a core/shell nanocrystal which is coated directly orindirectly with lipids, phospholipids, fatty acids, polynucleic acids,polyethylene glycol (PEG), primary antibodies, secondary antibodies,antibody fragments, protein or nucleic acid based aptamers, biotin,streptavidin, proteins, peptides, small organic molecules (e.g.,ligands), organic or inorganic dyes, precious or noble metal clusters.Specific examples of ligand coatings can include, but are not limitedto, amphiphilic polymer (AMP), bidentate thiols (i.e., DHLA), tridentatethiols, dipeptides, functionalized organophosphorous compounds (e.g.,phosphonic acids, phosphinic acids), etc.

Non-Blinking Nanoparticles

Provided herein are nanoparticles which exhibit modulated, reduced, orno intermittent (e.g., continuous, non-blinking) fluorescence.

In one aspect, the nanoparticle or populations thereof exhibitmodulated, reduced or non-detectable intermittent (e.g., continuous,etc.) fluorescence properties. The nanoparticles can have a stochasticblinking profile in a timescale which is shifted to very rapid blinkingor very slow or infrequent blinking relative to a nanoparticlepreviously described in the art (conventional nanoparticles aredescribed in the art as having on-time fractions of <0.2 in the best ofconditions examined). For example, the nanoparticles may blink on andoff on a timescale which is too rapid to be detected under the methodsemployed to study this behavior.

In one aspect the nanoparticle or populations thereof are photostable.The nanoparticles can exhibit a reduced or no photobleaching with longexposure to moderate to high intensity excitation source whilemaintaining a consistent spectral emission pattern.

In one aspect, the nanoparticle or populations thereof have aconsistently high quantum yield. For example, the nanoparticles can havea quantum yield greater than: about 10%, or about 20%, or about 30%, orabout 40%, or about 50%, or about 60%, or about 70% or about 80%.

As used herein, fluorescence (or Forster) resonance energy transfer(FRET) is a process by which a fluorophore (the donor) in an excitedstate transfers its energy to a proximal molecule (the acceptor) bynonradiative dipole-dipole interaction (Forster, T. “IntermolecularEnergy Migration and Fluorescence”, Ann. Phys., 2:55-75, 1948; Lakowicz,J. R., Principles of Fluorescence Spectroscopy, 2nd ed. Plenum, NewYork. 367-394., 1999).

FRET efficiency (E) can be defined as the quantum yield of the energytransfer transition, i.e. the fraction of energy transfer eventoccurring per donor excitation event. It is a direct measure of thefraction of photon energy absorbed by the donor which is transferred toan acceptor, as expressed in Equation 1: E=k_(ET)/k_(f)+k_(ET)+Σk_(i)where k_(ET) is the rate of energy transfer, k_(f) the radiative decayrate and the k_(i) are the rate constants of any other de-excitationpathway.

FRET efficiency E generally depends on the inverse of the sixth power ofthe distance r (nm) between the two fluorophores (i.e., donor andacceptor pair), as expressed in Equation 2: E=1/1+(r/R₀)⁶.

The distance where FRET efficiency is at 50% is termed R₀, also know asthe Forster distance. R₀ can be unique for each donor-acceptorcombination and can range from between about 5 nm to about 10 nm.Therefore, the FRET efficiency of a donor (i.e., nanoparticle) describesthe maximum theoretical fraction of photon energy which is absorbed bythe donor (i.e., nanoparticle) and which can then be transferred to atypical organic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).

In some embodiments, the disclosed nanoparticles are relatively small(i.e., <15 nm) and thus may be particularly well suited to be used as adonor or an acceptor in a FRET reaction. That is, some embodiments ofthe disclosed nanoparticles exhibit higher FRET efficiency thanconventional nanoparticles and thus are excellent partners (e.g., donorsor acceptors) in a FRET reaction.

“Quantum yield” as used herein refers to the emission efficiency of agiven fluorophore assessed by the number of times which a defined event,e.g., light emission, occurs per photon absorbed by the system. In otherwords, a higher quantum yield indicates greater efficiency and thusgreater brightness of the described nanoparticle or populations thereof.

Any suitable method can be used to measure quantum yield. In oneexample, quantum yield can be obtained using standard methods such asthose described in Casper et al (Casper, J. V.; Meyer, T. J. J. Am.Chem. Soc. 1983, 105, 5583) and can be analyzed relative to knownfluorophores chosen as appropriate for maximal overlap between standardemission and sample emission (e.g., fluorescein, Rhodamine 6G, Rhodamine101). Dilute solutions of the standard and sample can be matched ornearly matched in optical density prior to acquisition of absorbance andemission spectra for both. The emission quantum yield (ϕ_(em)) then canbe determined according to Equation 3:

$\phi_{em} = {{\phi_{em}^{\prime}\left( \frac{I}{I^{\prime}} \right)}\left( \frac{A^{\prime}}{A} \right)}$

where A and A′ are the absorbances at the excitation wavelength for thesample and the standard respectively and I and I′ are the integratedemission intensities for the sample and standard respectively. In thiscase ϕ′_(em) can be the agreed upon quantum yield for the standard.

Disclosed herein are fluorescent nanoparticles with superior and robustproperties which significantly expand the applications in whichnanoparticles are useful. These nanoparticles are superior andsurprisingly robust in that they are simultaneously stable, bright, andsensitive to environmental stimuli. Moreover, the disclosednanoparticles have limited or no detectable blinking (i.e., where thenanoparticle emits light non-intermittently when subject to excitation),are highly photostable, have a consistently high quantum yield, aresmall (e.g., ≤20 nm) and can act as a donor which undergoes FRET with asuitable acceptor moiety (e.g., fluorescent dyes, etc.). Thephotostability of these nanoparticles is reflected in their exhibitingreduced or no photobleaching (i.e., fading) behavior when subjected tomoderate to high intensity excitation for at least about 20 minutes.Additionally, the particles can remain substantially free fromphoto-induced color shifting.

Put another way, the nanoparticles can maintain a consistent spectralemission pattern (i.e., maintain the ability to fluoresce) even whenexposed to a large quantity of photons (i.e., moderate to high intensityexcitation) for a long period of time. This unique combination ofcharacteristics makes these types of nanoparticles sensitive tools forsingle molecule analysis and other sensitive high throughputapplications. Moreover, these properties make the nanoparticlesparticularly well suited for use as highly efficient donor fluorophoresin energy transfer reactions such as FRET reactions (i.e., high FRETefficiency) or other reactions as well as applications which require orare enhanced by greater response to the environment.

Without being bound to a particular theory, blinking or fluorescenceintermittency may arise during the nanoparticle charging process when anelectron is temporarily lost to the surrounding matrix (Auger ejectionor charge tunneling) or captured to surface-related trap states. Thenanoparticle is “on” or fluorescing when all of the electrons are intactand the particle is “neutral” and the particle is “off” or dark when theelectron is lost and the particle is temporarily (or in some casespermanently) charged. It is important to note that the completesuppression of blinking may not necessarily be required and in someinstances may not be desirable. Blinking which occurs on a timescalemuch shorter or much longer than the interrogation period for aparticular assay has relatively little impact on the performance of thesystem. Thus, nanoparticles and nanoparticle populations havingmodulated blinking properties, where blinking occurs on a very short orvery fast timescale relative to the assay interrogation periods are alsouseful and fall within the scope of the present disclosure. Localizationof timescale or simply pushing timescale to one side (e.g., to where theblinking is undetectable within the assay system) can providesubstantial benefit in application development.

The blinking behavior of the nanoparticles described herein can beanalyzed and characterized by any suitable number of parameters usingsuitable methodologies. The probability distribution function of the“on” and “off” blinking time durations (i.e., blinking behavior) can bedetermined using the form of an inverse power law. A value, alpha (α)can be calculated, wherein a represents an exponent in the power law. Asthe percentage of the population which is non-blinking increases, thevalue of α_(on) theoretically approaches zero. In conventionalnanoparticle populations previously described, α_(on) typically rangesfrom about 1.5 to about 2.5, under moderate to high excitation energy.

Most alpha calculations can use a predetermined threshold to determinethe “on” and “off” values of alpha-on and alpha-off (i.e., α_(on) andα_(off)). Typically, an alpha estimator which calculates the on/offthreshold for each dot individually can be employed. The data can berepresented by a plot of signal versus frequency, and typically appearsas a series of Gaussian distributions around the “off state” and one ormore “on states.” A log-log plot of frequency versus time for eachperiod of time that the dot is “on” provides a straight line having aslope of α_(on). The value of alpha-off (α_(off)) can be similarlydetermined.

In a specific example (the “TIRF example”), the fluorescentintermittency measurements can be made using a Total Internal ReflectionFluorescence (TIRF) microscope fitted with a 60× oil immersion objectivelens, using a dual view with a longpass filter on the acceptor side anda bandpass filter on the donor side. Using the TIRF setup, thenanoparticles were imaged at 30 Hz (33 ms), typically for 5 minutes, toproduce a movie showing the time and intensity of the emitted light foreach individual spot (corresponding to a single particle) within abinned frame which was 33 ms long; the intensity for each binned framecan be integrated. Each data set can be manually analyzed dot-by-dot,and aggregates and other artifacts were excluded. From the editedresults, the following parameters can be calculated: alpha-on(“α_(on)”); alpha-off (“α_(off)”); the percent on; longest on/longestoff; overlap scores; and the median values for each of these parameters.

In some aspects, provided herein is a nanoparticle or population thereofwhich has an α_(on) of less than about 1.5, α_(on) of less than about1.4, α_(on) of less than about 1.3, α_(on) of less than about 1.2, or anα_(on) of less than about 1.1, under moderate to high excitation energy.In some embodiments, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 98%, at least about 99% or more of the population has anα_(on) of less than about 1.5, α_(on) of less than about 1.4, α_(on) ofless than about 1.3, α_(on) of less than about 1.2, or α_(on) of lessthan about 1.1 for the time observed, under moderate to high excitationenergy. The observation time can be at least about 5 minutes, at leastabout 10 minutes, at least about 15 minutes, at least about 30 minutes,at least about 45 minutes, at least about 60 minutes, at least about 90minutes, at least about 120 minutes or more under moderate to highexcitation energy. Compositions comprising such a nanoparticle andpopulations thereof also are contemplated.

In some aspects, provided herein is a nanoparticle or a populationthereof having a stochastic blinking profile which is eitherundetectable or rare (e.g., no more than 1-2 events during theinterrogation period) over an observed timescale. In this case,“undetectable” encompasses the situation in which evidence might existfor ultra-fast blinking on a timescale which is faster than the binningtimescale (e.g., dimming and brightening from bin to bin) but there areno “off” events persisting for longer than the bin time. Therefore, insome embodiments, a nanoparticle or population thereof has a stochasticblinking profile which is undetectable for at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99% or more of the time observed, under moderate to highexcitation energy. In other embodiments, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 98%, at least about 99% or more ofthe individual nanoparticles in a population have a stochastic blinkingon a timescale which is undetectable for the time observed, undermoderate to high excitation energy. The timescale can be at least about5 minutes, at least about 10 minutes, at least about 15 minutes, atleast about 30 minutes, at least about 45 minutes, at least about 60minutes, at least about 90 minutes, at least about 120 minutes or moreunder moderate to high excitation energy.

In some aspects, the longest on and longest off values can relate to thelongest period of time a nanoparticle is observed to be in either the“on” or the “off” state. In particular, the longest on value can beimportant to determining the length of time and amount of data which maybe measured in a particular assay.

Thus, the blinking characteristics of the nanoparticles herein can alsobe characterized by their on-time fraction, which represents the (totalon-time)/(total experiment time). Under the TIRF example disclosedherein, the total on time can be determined by the total number offrames “on” multiplied by 33 ms, and the total experiment time is 5minutes. For example, the blinking properties of the disclosednanoparticles or populations thereof can be determined under continuousirradiation conditions using a 405 nm laser with an intensity of about 1watt per cm² during an experimental window of at least 5 minutes.

On-time fractions can be used to characterize the blinking behavior of asingle nanoparticle or of a population of nanoparticles. It is importantto note that the on-time fraction for a particular nanoparticle orpopulation of nanoparticles is a function of the specific conditionsunder which the percent of blinking or “non-blinking” nanoparticles isdetermined.

In some aspects, provided herein is a nanoparticle or population thereofhaving an on-time fraction of at least about 0.50, at least about 0.60,at least about 0.70, at least about 0.75, at least about 0.80, at leastabout 0.85, at least about 0.90, at least about 0.95, at least about0.96, at least about 0.97, at least about 0.98, or at least about 0.99or more, under moderate to high excitation energy. In some embodiments,a nanoparticle or populations thereof having a percent on-time of about98%, about 99% (i.e., on-time fraction of about 0.99) can be consideredto be “non-blinking,” under moderate to high excitation energy. In someembodiments, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more of the individual nanoparticlesin a population of nanoparticles can have an on-time fraction of atleast about 0.50, at least about 0.60, at least about 0.70, at leastabout 0.75, at least about 0.80, at least about 0.85, at least about0.90, at least about 0.95, at least about 0.96, at least about 0.97, atleast about 0.98, or at least about 0.99 or more, under moderate to highexcitation energy. The on-times of the nanoparticles are typically forat least about 5 minutes, at least about 10 minutes, at least about 15minutes, at least about 20 minutes, at least about 30 minutes, at leastabout 45 minutes, at least about 60 minutes, at least about 70 minutes,at least about 80 minutes, at least about 90 minutes, at least about 120minutes under moderate to high intensity excitation of the nanoparticleor nanoparticle population. Under one set of conditions, continuousirradiation with 405 nm laser with an approximate intensity of 1 wattper cm² was used to determine the stochastic blinking profile.

In some embodiments, nanoparticles which have a stochastic (i.e.,random) blinking profile in a timescale which shifts from very rapidblinking or very slow/infrequent blinking (relative to a nanoparticlepreviously described in the art) can be considered to have modulatedblinking properties. In some embodiments, these nanoparticles may blinkon and off on a timescale which is too rapid to be detected under themethods employed to study this behavior. Thus, certain nanoparticles caneffectively appear to be “always on” or to have on-time fractions ofabout 0.99, when in fact they flicker on and off at a rate too fast ortoo slow to be detected. Such flickering has relatively little impact onthe performance of a system, and for practical purposes suchnanoparticles can be considered to be non-blinking.

In some instances, the disclosed nanoparticles and populations thereofare not observed to blink off under the analysis conditions, and suchparticles can be assessed as “always on” (e.g., non-blinking). Thepercent of usable dots which are “always on” can be a useful way tocompare nanoparticles or populations of nanoparticles. However, adetermination of “always on” may mean that the “off” time wasinsufficient to provide enough a signal gap for accurate determinationand thus the value in the regime of particles is insufficient tocalculate. Even these “non-blinking” nanoparticles may flicker on andoff on a timescale which is not detected under the conditions used toassess blinking. For example, certain particles may blink on a timescalewhich is too fast to be detected, or they may blink very rarely, and, insome embodiments, such particles may also be considered to be“always-on” or non-blinking, as the terms are used herein.

In one aspect, provided herein is a nanoparticle or population thereofwhich demonstrate some fluctuation in fluorescence intensity. In someembodiments, the change in fluorescence intensity for the nanoparticleis less than about 5%, less than about 10%, less than about 20%, or lessthan about 25% of the nanoparticle or populations thereof at itsgreatest intensity, under moderate to high excitation energy. In someembodiments, such changes in fluorescence intensity of less than about5%, less than about 10%, less than about 20%, or less than about 25% ofthe highest intensity can occur in at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99% of the nanoparticles in the population, under moderate to highexcitation energy.

In some aspects, the nanoparticles with modulated, reduced or nointermittent (e.g., continuous, non-blinking) fluorescence providedherein can comprise of a core and a layered gradient shell. In someembodiments, the nanoparticle(s) disclosed herein can be comprised of ananocrystal core (e.g., CdSe, etc.), at least one inner (intermediate)shell layer (e.g., CdS, etc.), and at least one outer (external) shelllayer (e.g., ZnS, etc.). In some embodiments, the inner and/or outershell layers are each comprised of two or more discrete monolayers ofthe same material. In some embodiments, the largest dimension of thedisclosed nanoparticle(s) is less than about 15 nm. See for example, PCTApplication Serial No. PCT US/09/61951. See also PCT/US09/061951 andPCT/US09/061953 both filed on Oct. 23, 2009.

As discussed previously, the disclosed nanoparticles may be particularlywell suited for use as a donor or acceptor which undergoes FRET with asuitable complementary partner (donor or acceptor). A “FRET capable”nanoparticle refers to a nanoparticle which can undergo a measurableFRET energy transfer event with a donor or an acceptor moiety. In someembodiments, a FRET capable nanoparticle is one which has at least about25% efficiency in a FRET reaction.

Thus, in one aspect, a FRET capable fluorescent nanoparticle orpopulation thereof with modulated, reduced or non intermittent (e.g.,continuous, etc.) fluorescence is provided. In some embodiments, thenanoparticle is the donor in a FRET reaction. In some embodiments, thenanoparticle is the acceptor in the FRET reaction.

In some embodiments, the FRET capable non-blinking fluorescentnanoparticle(s) disclosed herein can comprise a core and a layeredgradient shell. In some embodiments, the FRET capable non-blinkingnanoparticle(s) disclosed herein can be comprised of a nanocrystal core(e.g., CdSe, etc.), at least one inner (intermediate) shell layer (e.g.,CdS, etc.), and at least one outer (external) shell layer (e.g., ZnS,etc.). In some embodiments, the inner and/or outer shell layers are eachcomprised of two or more discrete monolayers of the same material. Insome embodiments, the largest dimension of the disclosed FRET capablenanoparticle(s) is less than about 15 nm.

In some embodiments, the nanoparticle or population thereof has a FRETefficiency of at least about 20%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or greater.

In some embodiments, at least about 30%, at least about 40%, at leastabout 50%, at least about 60% at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 98%, at least about99% or more of the individual nanoparticles in the population have aFRET efficiency of at least about 20%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99% or more.

In some embodiments, the FRET efficiency of the disclosed nanoparticleor population thereof can be maintained for at least about the first10%, at least about the first 20%, at least about the first 30%, atleast about the first 40%, at least about the first 50%, at least aboutthe first 60%, at least about the first 70%, at least about the first80%, at least about the first 90% or more of the total emitted photonsunder conditions of moderate to high excitation.

As discussed above, the nanoparticle(s) provided herein can beconsidered to be surprisingly photostable. In particular, thenanoparticle and populations described herein can be photostable over anextended period of time while maintaining the ability to effectivelyparticipate in energy transfer (i.e., FRET) reactions. The disclosednanoparticles can be stable under high intensity conditions involvingprolonged or continuous irradiation over an extended period of time froma moderate to high excitation source.

Thus, in one aspect, provided herein is a non-blinking fluorescentnanoparticle and population thereof which is photostable.

In some embodiments, the disclosed photostable nanoparticle andpopulation thereof can have an emitted light or energy intensitysustained for at least about 10 minutes and does not decrease by morethan about 20% of maximal intensity achieved during that time. Further,these nanoparticles and populations thereof can have a wavelengthspectrum of emitted light which does not change more than about 10% uponprolonged or continuous exposure to an appropriate energy source (e.g.irradiation).

In one embodiment, the photostable nanoparticles disclosed herein canremain photostable under moderate to high intensity excitation from atleast about 10 minutes to about 2 hours. In another embodiment, thephotostable nanoparticles disclosed herein can remain photostable undermoderate to high intensity excitation from at least about 10 minutes toabout 10 hours. In still another embodiment, the photostablenanoparticles disclosed herein can remain photostable under moderate tohigh from about 10 minutes to about 48 hours. However, it should beappreciated, that these are just example photostable times for thedisclosed nanoparticles, in practice the nanoparticles can remainphotostable for longer periods of time depending on the particularapplication.

It should be appreciated that nanoparticles which are photostable overlonger timescales in combination with moderate to high excitation energysources are well suited for more sensitive and broad-rangingapplications such as the real-time monitoring of single moleculesinvolving FRET. That is, the nanoparticle and population thereofdescribed herein can be photostable over an extended period of timewhile maintaining the ability to effectively participate in energytransfer (i.e., FRET) reactions, which makes the subject nanoparticlesparticularly useful for many applications involving the real-timemonitoring of single molecules. As such, in some embodiments thephotostable nanoparticles disclosed herein have FRET efficiencies of atleast about 20%.

In some embodiments, the disclosed nanoparticles are stable uponprolonged or continuous irradiation (under moderate to high excitationrate) in which they do not exhibit significant photo-bleaching on thetimescales indicated. Photobleaching can result from the photochemicaldestruction of a fluorophore (and can be characterized by thenanoparticles losing the ability to produce a fluorescent signal) by thelight exposure or excitation source used to stimulate the fluorescence.Photobleaching can complicate the observation of fluorescent moleculesin microscopy and the interpretation of energy transfer reactionsbecause the signals can be destroyed or diminished increasingly astimescales for the experiment increase or the energy intensityincreases.

Photobleaching can be assessed by measuring the intensity of the emittedlight or energy for a nanoparticle or nanoparticle population using anysuitable method. In some embodiments, the intensity of emitted light orenergy from the disclosed nanoparticle or population thereof does notdecrease by more than about 20% (and in some embodiments, not more thanabout 10%) upon prolonged or continuous irradiation (under moderate tohigh excitation rate). In some embodiments, the intensity of emittedlight from the disclosed nanoparticle or population thereof does notdecrease by more than about 20%, about 15%, about 10%, about 5% or lessupon irradiation from about 10 minutes, about 20 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2hours, about 3 hours to about 4 hours, under moderate to high excitationenergy.

In some embodiments, the photostable nanoparticles provided hereinfurther demonstrate enhanced stability in which they exhibit a reductionin or absence of spectral shifting during prolonged excitation. In theconventional nanoparticles previously described in the art, increasedexposure to an excitation source—whether via increase time orpower—results in a spectral shift of the wavelength emission wavelengthprofile of a nanoparticle and populations thereof from a longerwavelength to an increasingly shorter wavelength. Such spectral shiftingof emission wavelength represents a significant limitation as preciseresolution of emission spectra is required for applications whichrequire rapid detection, multi-color analysis, and the like. Shifting ofany significance then requires that the wavelength emissions used in anassay be sufficiently separated to permit resolution, thus reducing thenumber of colors available as well as increasing signal to noise ratioto an unacceptable level as the initial spectral profile cannot berelied upon once spectral shifting begins. Such shifting may requireshortened observation times or use of fluorophores with widely separatedemission spectra. The nanoparticles provided herein have little to nospectral shift, particularly over extended periods of excitation.

Wavelength emission spectra can be assessed by any suitable method. Forexample, spectral characteristics of nanoparticles can generally bemonitored using any suitable light-measuring or light-accumulatinginstrumentation. Examples of such instrumentation are CCD(charge-coupled device) cameras, video devices, CIT imaging, digitalcameras mounted on a fluorescent microscope, photomultipliers,fluorometers and luminometers, microscopes of various configurations,and even the human eye. The emission can be monitored continuously or atone or more discrete time points. The photostability and sensitivity ofnanoparticles allow recording of changes in electrical potential overextended periods of time.

Thus, in some embodiments, the photostable nanoparticle and populationthereof has a wavelength spectrum of emitted light which does not changemore than about 10% upon prolonged or continuous exposure to anappropriate energy source (e.g. irradiation) over about 4 minutes toabout 10 minutes, under moderate to high excitation energy. In someembodiments, the wavelength emission spectra does not change more thanabout 5%, more than about 10%, more than about 20% over 10 minutes,about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes,about 90 minutes, about 2 hours, about 3 hours to about 4 hours.

It should be appreciated that there can be various other objectiveindicia of nanoparticle photostability. For example, a nanoparticle canbe classified as photostable when the nanoparticle, under moderate tohigh excitation, emits about 1,000,000 to about 100,000,000 photons ormore preferably about 100,000,001 to about 100,000,000,000 photons oreven more preferably more than about 100,000,000,000 photons beforebecoming non-emissive (i.e., bleached).

A nanoparticle with modulated, reduced or no fluorescent intermittency(e.g., continuous, non-blinking, etc.); reduced or absent spectralshifting; low to no photobleaching; high quantum yield; and sufficientFRET efficiency can be of any suitable size. Typically, it is sized toprovide fluorescence in the UV-visible portion of the electromagneticspectrum as this range is convenient for use in monitoring biologicaland biochemical events in relevant media. The disclosed nanoparticle andpopulation thereof can have any combination of the properties describedherein.

Thus, in some embodiments the nanoparticle or population thereof hasmodulated or no blinking, are photostable (e.g., limited or nophotobleaching, limited or no spectral shift), has high quantum yield,have high FRET efficiency, has a diameter of less than about 15 nm, isspherical or substantially spherical shape, or any combination of allthese properties as described herein.

Likewise, in some embodiments, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or more of theindividual nanoparticles in a population of nanoparticles have modulatedor no blinking, are photostable (e.g., limited or no photobleaching,limited or no spectral shift), have high quantum yield, have high FRETefficiency, have diameters of less than about 15 nm, are spherical orsubstantially spherical shape, or any combination of or all of theseproperties as described herein.

In one aspect, the FRET capable, non-blinking and/or photostablenanoparticle or population thereof provided herein has a maximumdiameter of less than about 20 nm. In some embodiments, thenanoparticle(s) can be less than about 15 nm, less than about 10 nm,less than about 8 nm, less than about 6 nm, less than about 5 nm, lessthan about 4 nm, less than about 3 nm or less in its largest diameterwhen measuring the core/shell structure. Any suitable method may be usedto determine the diameter of the nanoparticle(s). The nanoparticle(s)provided herein can be grown to the desired size using any of themethods disclosed herein. In some embodiments, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or moreof the individual members of a population of nanoparticles have maximumdiameters (when measuring the core, core/shell or core/shell/ligandstructure) which are less than about 20 nm, less than about 15 nm, lessthan about 10 nm, less than about 8 nm, less than about 6 nm, less thanabout 5 nm, less than about 4 nm, less than about 3 nm or less.

The FRET capable, non-blinking and/or photostable nanoparticle(s)provided herein and populations thereof can be spherical orsubstantially spherical. In some embodiments, a substantially sphericalnanoparticle can be one where any two radius measurements do not differby more than about 10%, about 8%, about 5%, about 3% or less. In someembodiments, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more of the individual members of apopulation of nanoparticles are spherical or substantially spherical.

Nanoparticles can be synthesized in shapes of different complexity suchas spheres, rods, discs, triangles, nanorings, nanoshells, tetrapods,nanowires and so on. Each of these geometries can have distinctiveproperties: spatial distribution of the surface charge, orientationdependence of polarization of the incident light wave, and spatialextent of the electric field. In some embodiments, the nanoparticles aresubstantially spherical or spheroidal.

For embodiments where the nanoparticle is not spherical or spheroidal,e.g. rod-shaped, it may be from about 1 to about 15 nm, from about 1 nmto about 10 nm, or 1 nm to about 5 nm in its smallest dimension. In somesuch embodiments, the nanoparticles may have a smallest dimension ofabout 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm,about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 25 nm, about30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm and rangesbetween any two of these values.

The single-color preparation of the nanoparticles disclosed herein canhave individual nanoparticles which are of substantially identical sizeand shape. Thus, in some embodiments, the size and shape between theindividual nanoparticles in a population of nanoparticles vary by nomore than about 20%, no more than about 15%, no more than about 10%, nomore than about 8%, no more than about 6%, no more than about 5%, nomore than about 4%, no more than about 3% or less in at least onemeasured dimension. In some embodiments, disclosed herein is apopulation of nanoparticles, where at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, andideally about 100% of the particles are of the same size. Size deviationcan be measured as root mean square (“rms”) of the diameter, with thepopulation having less than about 30% rms, preferably less than about20% rms, more preferably less than about 10% rms. Size deviation can beless than about 10% rms, less than about 9% rms, less than about 8% rms,less than about 7% rms, less than about 6% rms, less than about 5% rms,less than about 3% rms, or ranges between any two of these values. Sucha collection of particles is sometimes referred to as being a“monodisperse” population.

The color (emitted light) of a nanoparticle can be “tuned” by varyingthe size and composition of the particle. Nanoparticles as disclosedherein can absorb a wide spectrum of wavelengths, and emit a relativelynarrow wavelength of light. The excitation and emission wavelengths aretypically different, and non-overlapping. The nanoparticles of amonodisperse population may be characterized in that they produce afluorescence emission having a relatively narrow wavelength band.Examples of emission widths include less than about 200 nm, less thanabout 175 nm, less than about 150 nm, less than about 125 nm, less thanabout 100 nm, less than about 75 nm, less than about 60 nm, less thanabout 50 nm, less than about 40 nm, less than about 30 nm, less thanabout 20 nm, and less than about 10 nm. In some embodiments, the widthof emission is less than about 60 nm full width at half maximum (FWHM),or less than about 50 nm FWHM, and sometimes less than about 40 nm FWHM,less than about 30 nm FWHM or less than about 20 nm FWHM. In someembodiments, the emitted light preferably has a symmetrical emission ofwavelengths.

The emission maxima of the disclosed nanoparticle and population thereofcan generally be at any wavelength from about 200 nm to about 2,000 nm.Examples of emission maxima include about 200 nm, about 400 nm, about600 nm, about 800 nm, about 1,000 nm, about 1,200 nm, about 1,400 nm,about 1,600 nm, about 1,800 nm, about 2,000 nm, and ranges between anytwo of these values.

As discussed previously, the disclosed nanoparticle or populationsthereof can comprise a core and a layered shell, wherein the shellincludes at least one inner (intermediate) shell layer comprising afirst shell material and at least one outer (external) shell layercomprising a second shell material, and wherein the layered shell issubstantially uniform in coverage around the core and is substantiallyfree of defects.

Thus, in one aspect, the nanoparticle or population thereof comprises acore (M¹Y) and a layered shell, wherein the shell comprises m innershell monolayers comprising a first shell material (M¹X)_(m) and n outershell monolayers comprising a second shell material (M²X)_(n), wherein Mcan be a metal atom and X can be a non-metal atom, each of m and n isindependently an integer from 1 to 10, and the layered shell issubstantially uniform in coverage around the core and is substantiallyfree of defects. In specific embodiments, the sum of m+n is 3-20, or5-14, or 6-12, or 7-10.

In certain embodiments, the disclosed nanoparticles can further compriseone or more additional shell layers between the at least one inner shelllayer and the at least one outer shell layer.

In some embodiments, the nanoparticle core and population thereof canhave a first bandgap energy and the first shell material can have asecond bandgap energy, wherein the second bandgap energy can be greaterthan the first bandgap energy.

In a further aspect, provided herein is a nanoparticle or populationthereof comprising a core and a layered shell, wherein the shellcomprises sequential monolayers comprising an alloyed multi-componentshell material of the form M¹ _(x)M² _(y)X, where M¹ and M² can be metalatoms and X can be a non metal atom, where the composition becomessuccessively enriched in M² as the monolayers of shell material aredeposited, where x and y represent the ratio of M¹ and M² in the shellmaterial, and wherein the monolayered shell is substantially uniform incoverage around the core and is substantially free of defects. In someembodiments, the layered shell sometimes has about 3-20 monolayers ofshell material, sometimes about 5-14 monolayers of shell material,sometimes about 6-12 monolayers of shell material, or sometimes about7-10 monolayers of shell material.

In one aspect, provided herein is a nanoparticle or population thereofcomprising a core and a layered shell having a gradient potential,wherein the shell comprises at least one inner shell layer and at leastone outer shell layer, and wherein the layered shell is substantiallyuniform in coverage around the core and is substantially free ofdefects.

The layered shell may be engineered such that the sequential monolayersare selected to provide a gradient potential from the nanoparticle coreto the outer surface of the nanoparticle shell. The steepness of thepotential gradient may vary depending on the nature of the shellmaterials selected for each monolayer or group of monolayers. Forexample, a nanoparticle comprising several sequential monolayers of thesame shell material may reduce the potential through a series of steps,while a more continuous gradient may be achievable through the use ofsequential monolayers of a multi-component alloyed shell material. Insome embodiments, both single component and multi-component shellmaterials may be applied as different monolayers of a multi-layer shellon a nanoparticle.

The nanoparticles can be synthesized as disclosed to the desired size bysequential, controlled addition of materials to build and/or applymonolayers of shell material to the core. This is in contrast toconventional methods of adding shells where materials (e.g., diethylzincand bis(trimethylsilyl)sulfide) are added together. Sequential additionpermits the formation of thick (e.g., >2 nm) relatively uniformindividual shells (e.g., uniform size and depth) on a core. The layeradditions generally require the addition of an appropriate amount of theshell precursors to form a single monolayer, based on the starting sizeof the underlying core. This means that as each monolayer of shellmaterial is added, a new “core” size must be determined by taking theprevious “core” size and adding to it the thickness of just-added shellmonolayer. This leads to a slightly larger volume of the following shellmaterial needing to be added for each subsequent monolayer of shellmaterial being added.

Each monolayer of shell material can be independently selected, and maybe made up of a single component, or may comprise a multi-component(e.g., alloyed, etc.) shell material. In some embodiments, it issuitable to apply one or more sequential monolayers of a first shellmaterial, followed by one or more sequential monolayers of a secondshell material. This approach allows the deposition of at least oneinner shell layer of a material having a bandgap and lattice sizecompatible with the core, followed by the deposition of at least oneouter shell layer of a material having a bandgap and lattice sizecompatible with the inner shell layer. In some embodiments, multiplesequential monolayers of a single shell material can be applied toprovide a uniform shell of a desired number of monolayers of a singleshell material; in these embodiments, the first and second shellmaterials are the same. In other embodiments, sequential monolayers ofan alloyed shell material are applied, where the ratio of the componentsvaries such that the composition becomes successively enriched in onecomponent of the multi-component mixture as the successive monolayers ofshell material are deposited.

In some embodiments, the layered shell can be about 3-20 monolayers ofshell material thick, sometimes about 5-14 monolayers of shell materialthick, sometimes about 6-12 monolayers of shell material thick orsometimes about 7-10 monolayers of shell material thick. In someembodiments, at least one inner shell layer can be comprised of about3-5 monolayers, sometimes about 3-7 monolayers, of the first shellmaterial. In other embodiments, at least one outer shell layer can becomprised of about 3-5 monolayers, sometimes about 3-7 monolayers, ofthe second shell material. In some embodiments, the inner shell layercan be at least 3 monolayers thick; in other embodiments, the outershell layer can be at least 3 monolayers thick. The individualmonolayers can be formed by the controlled, sequential addition of thelayer materials methods described herein. The monolayers may not alwaysbe completely distinct as they may, in some embodiments, be a latticingbetween the surfaces of contacting monolayers.

In certain embodiments, provided herein are nanoparticles having athick, uniform, layered shell, as described herein, wherein the corecomprises CdSe, the at least one inner shell layer comprises CdS, andthe at least one outer shell layer comprises ZnS. In a particularembodiment, provided herein is a nanoparticle or population thereofhaving a CdSe core and a layered shell comprising 4CdS+3.5ZnS layers. Insome embodiments, provided herein is a nanoparticle which consistsessentially of CdSe/4CdS-3.5ZnS.

Also disclosed herein are methods of making a nanoparticle andpopulation thereof with modulated, reduced or no fluorescenceintermittency or “blinking”. These nanoparticles can be small,photostable, bright, highly FRET efficient or some combination thereof.These nanoparticles can have a multi-shell layered core achieved by asequential shell material deposition process, whereby one shell materialis added at a time, to provide a nanoparticle having a substantiallyuniform shell of desired thickness which is substantially free ofdefects.

In one aspect, provided herein is a method for making a nanoparticle orpopulation thereof with modulated, reduced or no fluorescenceintermittency, comprising: providing a mixture comprising a core and atleast one coordinating solvent; adding a first inner shell precursoralternately with a second inner shell precursor in layer additions, toform an inner shell layer which is a desired number of layers thick; andadding a first outer shell precursor alternately with a second outershell precursor in layer additions, to form an outer shell layer whichis a desired number of layers thick. If the coordinating solvent of isnot amine, the method further comprises an amine in.

In some embodiments, the mixture can be heated to a temperature which issuitable for shell formation before and/or after every sequentialaddition of a shell precursor. In some embodiments, the shell issubstantially uniform in coverage around the core and is substantiallyfree of defects. In some embodiments, the resulting nanoparticles have adiameter of less than about 15 nm. In other embodiments, thenanoparticles have a diameter of between about 6 nm to about 10 nm. Thenanoparticles made by this method can have quantum yields greater thanabout 80%. The nanoparticle made by this method can have on-timefractions (i.e., ratio of the time which nanoparticle emission is turned“on” when the nanoparticle is excited) of greater than about 0.80 (undermoderate to high excitation energy).

In another aspect, provided herein is a method for making a FRET capablenanoparticle and populations thereof with modulated, reduced or nofluorescence intermittency, comprising: (a) providing a mixturecomprising a plurality of nanocrystal cores and at least onecoordinating solvent; (b) adding a first intermediate shell precursoralternately with a second intermediate shell precursor in layeradditions to form an intermediate shell layer on each of the pluralityof nanocrystal cores, wherein the intermediate shell layer is comprisedof more than one monolayer; (c) adding a first external shell precursoralternately with a second external shell precursor in layer additions toform an external shell layer on each of the plurality of nanocrystalcores, wherein the external shell layer is disposed on top of theintermediate shell layer and is comprised of more than one monolayer;(d) adding an aqueous solution comprising a hydrophilic ligand; and (e)maintaining the mixture under conditions which cause the plurality ofnanocrystals to migrate into an aqueous phase. If the coordinatingsolvent is not an amine, at least one amine can be included in step (a).In some embodiments, the resulting population of FRET capablenon-blinking nanoparticles has a α_(on) value which is less than about1.4. In other embodiments, the resulting population of FRET capablenon-blinking nanoparticles has an on-time fraction of least about 0.8(under moderate to high excitation energy). In some embodiments, theresulting population of FRET capable non-blinking nanoparticles hasdiameters which are less than about 15 nm. In some embodiments, theresulting population of FRET capable non-blinking nanoparticles has aFRET efficiency of at least 20%. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a quantumyield of at least about 40%.

In some embodiments, the methods disclosed above utilize a one step or atwo step ligand exchange process to replace the hydrophobic ligands onthe nanoparticles with hydrophilic ligands to cause the plurality ofnanocrystals to migrate into the aqueous phase. See PCT ApplicationSerial No. PCT/US09/053,018 and PCT/US09/059,456 which are expresslyincorporated herein by reference as if set forth in full.

In another aspect, provided herein is a method for making a FRET capablenanoparticle and populations thereof with modulated, reduced or nofluorescence intermittency, comprising: providing a mixture comprising aplurality of nanocrystal cores, functionalized organophosphorous-basedhydrophilic ligands and at least one coordinating solvent; adding afirst intermediate shell precursor alternately with a secondintermediate shell precursor in layer additions to form an intermediateshell layer on each of the plurality of nanocrystal cores; and adding afirst external shell precursor alternately with a second external shellprecursor in layer additions to form an external shell layer on each ofthe plurality of nanocrystal cores. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has an α_(on)value which is less than about 1.4. In other embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has an on-timefraction of least about 0.8. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has diameterswhich are less than about 15 nm. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a FRETefficiency of at least 20%. In some embodiments, the resultingpopulation of FRET capable non-blinking nanoparticles has a quantumyield of at least about 40%.

In some embodiments, the functionalized organophosphorous-basedhydrophilic ligands are multi-functional surface ligands which include aphosphonate/phosphinate nanocrystal binding center, a linker, and afunctional group, which imparts functionality on the nanocrystal. Asused herein the term “functional group” may refer to a group whichaffects reactivity, solubility, or both reactivity and solubility whenpresent on a multi-functional surface ligand. Embodiments can include awide variety of functional groups which can impart various types offunctionality on the nanocrystal including hydrophilicity,water-solubility, or dispersibility and/or reactivity, and thefunctionality may generally not include only hydrophobicity or onlysolubility in organic solvents without increasing reactivity. Forexample, a functional group which is generally hydrophobic but whichincreases reactivity such as an alkene or alkyne and certain esters andethers can be encompassed by embodiments, whereas alkyl groups, which donot generally impart reactivity but increase hydrophobicity may beexcluded.

In certain embodiments, the FRET capable and non-blinking nanoparticlesproduced by the disclosed methods may be coated with ligands whichimpart water solubility and/or reactivity on the nanoparticle obviatingthe need for ligand replacement. Without wishing to be bound by theory,eliminating ligand replacement may provide more consistent thermodynamicproperties, which may lead to reduction in variability of coating andless loss of quantum yield, among other improvements in the propertiesof nanoparticles produced by the methods embodied herein. Eliminatingligand replacement may also allow for the production of nanoparticleshaving a wide variety of functional groups associated with the coating.In particular, while ligand replacement is generally limited toproduction of nanoparticles having amine and/or carboxylic acidfunctional groups, in various embodiments, the skilled artisan maychoose among numerous functional groups when preparing themulti-functional ligands and may, therefore, generate nanoparticleswhich provide improved water-solubility or water-dispersity and/orsupport improved crosslinking and/or improved reactivity with cargomolecules. See PCT Application Serial No. PCT/US09/059117 which isexpressly incorporated herein by reference as if set forth in full.

In another aspect, provided herein is a method of making a nanoparticleor population thereof comprising a core and a layered gradient shell,wherein the shell comprises an multi-component (e.g., alloy, etc.) shellmaterial of the form M¹ _(x)M² _(y)X, where x and y represent the ratioof M¹ and M² in the shell material. The method comprising: (a) providinga mixture comprising a core, at least one coordinating solvent; (b)heating said mixture to a temperature suitable for formation of theshell layer; and (c) adding a first inner shell precursor comprising M¹_(x) and M² _(y) alternately with a second inner shell precursorcomprising X in layer additions, wherein the ratio of y to x graduallyincreases in sequential layer additions, such that the shell layersbecomes successively enriched in M², to form a layered gradient shellwhich is a desired number of monolayers thick. If the coordinatingsolvent is not an amine, at least one amine can be included in step (a).

In one embodiment, the method described above provides a nanoparticlehaving a layered gradient shell, wherein the core comprises CdSe and theshell comprises sequential layers of Cd_(x)Zn_(y)S, where the ratio of yto x increases gradually from the innermost shell layer to the outermostshell layer, to provide a layered gradient shell with a finely gradedpotential. In some such embodiments, the outermost shell layer isessentially pure ZnS. In some embodiments, the percent of Zn in thegradient shell varies from less than about 10% at the innermost shelllayer to greater than about 80% at the outermost shell layer.

Typically, the heating steps in the disclosed methods are conducted at atemperature within the range of about 150-350° C., more preferablywithin the range of about 200-300° C. In some embodiments, thetemperature suitable for formation of at least one inner shell layer isabout 215° C. In some embodiments, the temperature suitable forformation of at least one outer shell layer is about 245° C. It isunderstood that the above ranges are merely exemplary and are notintended to be limiting in any manner as the actual temperature rangesmay vary, dependent upon the relative stability of the precursors,ligands, and solvents. Higher or lower temperatures may be appropriatefor a particular reaction. The determination of suitable time andtemperature conditions for providing nanoparticles is within the levelof skill in the art using routine experimentation.

It can be advantageous to conduct the nanoparticle-forming reactionsdescribed herein with the exclusion of oxygen and moisture. In someembodiments the reactions are conducted in an inert atmosphere, such asin a dry box. The solvents and reagents are also typically rigorouslypurified to remove moisture and oxygen and other impurities, and aregenerally handled and transferred using methods and apparatus designedto minimize exposure to moisture and/or oxygen. In addition, the mixingand heating steps can be conducted in a vessel which is evacuated andfilled and/or flushed with an inert gas such as nitrogen. The fillingcan be periodic or the filling can occur, followed by continuousflushing for a set period of time.

In some embodiments, the at least one coordinating solvent comprises atrialkylphosphine, a trialkylphosphine oxide, a phosphonic acid, or amixture of these. Sometimes, the at least one coordinating solventcomprises TOP, TOPO, TDPA, OPA or a mixture of these. The solvent forthese reactions often comprises a primary or secondary amine, forexample, decylamine, hexadecylamine, or dioctylamine. In someembodiments, the amine is decylamine. In some embodiments, the firstinner shell precursor is Cd(OAc)₂ and the second inner shell precursoris bis(trimethylsilyl)sulfide (TMS2S). Sometimes, the first and secondinner shell precursors are added as a solution in TOP. In someembodiments, the first outer shell precursor is Et2Zn and the secondinner shell precursor is TMS2S. Sometimes, the first and second outershell precursors are added as a solution in TOP.

In certain embodiments, the disclosed nanoparticles may be preparedusing the method described herein to build a layered CdS—ZnS shell on aCdSe quantum size core. The shells for these materials can have varyingnumbers of layers of CdS and ZnS. Prototypical materials containing aCdSe core and approximately 4 monolayers CdS and 3.5 monolayers of ZnS(the final 0.5 monolayer is essentially pure Zn), or a CdSe core and 9monolayers CdS and 3.5 monolayers of ZnS were prepared as described inthe examples.

In some embodiments, for either the inner or outer layer, or both, lessthan a full layer of the appropriate first shell precursor can be addedalternately with less than a full layer of the appropriate second shellprecursor, so the total amount of the first and second shell precursorrequired is added in two or more portions. Sometimes, the portion isabout 0.25 monolayers of shell material, so that the 4 portions of 0.25monolayer of first shell precursor are added alternately with 4 portionsof 0.25 monolayer of second shell precursor; sometimes the portion isabout 0.5 monolayers of shell material, and sometimes about 0.75monolayers of shell material.

Examples of compounds useful as the first precursor can include, but arenot limited to: organometallic compounds such as alkyl metal species,salts such as metal halides, metal acetates, metal carboxylates, metalphosphonates, metal phosphinates, metal oxides, or other salts. In someembodiments, the first precursor provides a neutral species in solution.For example, alkyl metal species such as diethylzinc (Et₂Zn) or dimethylcadmium are typically considered to be a source of neutral zinc atoms(Zn⁰) in solution. In other embodiments, the first precursor provides anionic species (i.e., a metal cation) in solution. For example, zincchloride (ZnCl₂) and other zinc halides, zinc acetate (Zn(OAc)₂) andzinc carboxylates are typically considered to be sources of Zn²⁺ cationsin solution.

By way of example only, suitable first precursors providing neutralmetal species include dialkyl metal sources, such as dimethyl cadmium(Me₂Cd), diethyl zinc (Et₂Zn), and the like. Suitable first precursorsproviding metal cations in solution include, e.g., cadmium salts, suchas cadmium acetate (Cd(OAc)₂), cadmium nitrate (Cd(NO₃)₂), cadmium oxide(CdO), and other cadmium salts; and zinc salts such as zinc chloride(ZnCl₂), zinc acetate (Zn(OAc)₂), zinc oleate (Zn(oleate)₂), zincchloro(oleate), zinc undecylenate, zinc salicylate, and other zincsalts. In some embodiments, the first precursor is salt of Cd or Zn. Insome embodiments, it is a halide, acetate, carboxylate, or oxide salt ofCd or Zn. In other embodiments, the first precursor is a salt of theform M(O₂CR)X, wherein M is Cd or Zn; X is a halide or O₂CR; and R is aC4-C24 alkyl group which is optionally unsaturated. Other suitable formsof Groups 2, 12, 13 and 14 elements useful as first precursors are knownin the art.

Precursors useful as the “second” precursor in the disclosed methodsinclude compounds containing elements from Group 16 of the PeriodicTable of the Elements (e.g., S, Se, Te, and the like), compoundscontaining elements from Group 15 of the Periodic Table of the Elements(N, P, As, Sb, and the like), and compounds containing elements fromGroup 14 of the Periodic Table of the Elements (Ge, Si, and the like).Many forms of the precursors can be used in the disclosed methods. Itwill be understood that in some embodiments, the second precursor willprovide a neutral species in solution, while in other embodiments thesecond precursor will provide an ionic species in solution.

When the first precursor comprises a metal cation, the second precursorcan provide an uncharged (i.e., neutral) non-metal atom in solution. Infrequent embodiments, when the first precursor comprises a metal cation,the second precursor contributes a neutral chalcogen atom, most commonlyS⁰, Se⁰ or Te⁰.

Suitable second precursors for providing a neutral chalcogen atominclude, for example, elemental sulfur (often as a solution in an amine,e.g., decylamine, oleylamine, or dioctylamine, or an alkene, such asoctadecene), and tri-alkylphosphine adducts of S, Se and Te. Suchtrialkylphosphine adducts are sometimes described herein as R3P=X,wherein X is S, Se or Te, and each R is independently H, or a C1-C24hydrocarbon group which can be straight-chain, branched, cyclic, or acombination of these, and which can be unsaturated. Exemplary secondprecursors of this type include tri-n (butylphosphine)selenide (TBP=Se),tri-n-(octylphosphine)selenide (TOP=Se), and the corresponding sulfurand tellurium reagents, TBP=S, TOP=S, TBP=Te and TOP=Te. These reagentsare frequently formed by combining a desired element, such as Se, S, orTe with an appropriate coordinating solvent, e.g., TOP or TBP.Precursors which provide anionic species under the reaction conditionsare typically used with a first precursor which provides a neutral metalatom, such as alkylmetal compounds and others described above or knownin the art.

In some embodiments, the second precursor provides a negatively chargednon-metal ion in solution (e.g., S-2, Se-2 or Te-2). Examples ofsuitable second precursors providing an ionic species include silylcompounds such as bis(trimethylsilyl)selenide ((TMS)₂Se),bis(trimethylsilyl)sulfide ((TMS)₂S) and bis(trimethylsilyl)telluride((TMS)₂Te). Also included are hydrogenated compounds such as H2Se, H2S,H2Te; and metal salts such as NaHSe, NaSH or NaHTe. In this situation,an oxidant can be used to oxidize a neutral metal species to a cationicspecies which can react with the anionic precursor in a ‘matched’reaction, or an oxidant can be used increase the oxidation state of theanionic precursor to provide a neutral species which can undergo a‘matched’ reaction with a neutral metal species.

Other exemplary organic precursors are described in U.S. Pat. Nos.6,207,229 and 6,322,901 to Bawendi et al., and synthesis methods usingweak acids as precursor materials are disclosed by Qu et al., (2001),Nano Lett., 1(6):333-337, the disclosures of each of which areincorporated herein by reference in their entirety.

Both the first and the second precursors can be combined with anappropriate solvent to form a solution for use in the disclosed methods.The solvent or solvent mixture used to form a first precursor solutionmay be the same or different from that used to form a second precursorsolution. Typical coordinating solvents include alkyl phosphines, alkylphosphine oxides, alkyl phosphonic acids, alkyl phosphinic acids, orcarboxylic acid containing solvents, or mixtures of these.

Suitable reaction solvents include, by way of illustration and notlimitation, hydrocarbons, amines, alkyl phosphines, alkyl phosphineoxides, carboxylic acids, ethers, furans, phosphoacids, pyridines andmixtures thereof. The solvent may actually comprise a mixture ofsolvents, often referred to in the art as a “solvent system”. In someembodiments, the solvent comprises at least one coordinating solvent. Insome embodiments, the solvent system comprises a secondary amine and atrialkyl phosphine (e.g., TBP or TOP) or a trialkylphosphine oxide(e.g., TOPO). If the coordinating solvent is not an amine, an amine canbe included.

A coordinating solvent might be a mixture of an essentiallynon-coordinating solvent such as an alkane and a ligand as definedbelow.

Suitable hydrocarbons include alkanes, alkenes and aromatic hydrocarbonsfrom 10 to about 30 carbon atoms; examples include octadecene andsqualane. The hydrocarbon may comprise a mixture of alkane, alkene andaromatic moieties, such as alkylbenzenes (e.g., mesitylene).

Suitable amines include, but are not limited to, monoalkylamines,dialkylamines, and trialkylamines, for example dioctylamine, oleylamine,decylamine, dodecylamine, hexyldecylamine, and so forth. Alkyl groupsfor these amines typically contain about 6-24 carbon atoms per alkyl,and can include an unsaturated carbon-carbon bond, and each aminetypically has a total number of carbon atoms in all of its alkyl groupscombined of about 10-30 carbon atoms.

Exemplary alkyl phosphines include, but are not limited to, the trialkylphosphines, tri-n-butylphosphine (TBP), tri-n-octylphosphine (TOP), andso forth. Alkyl groups for these phosphines contain about 6-24 carbonatoms per alkyl, and can contain an unsaturated carbon-carbon bond, andeach phosphine has a total number of carbon atoms in all of its alkylgroups combined of about 10-30 carbon atoms.

Suitable alkyl phosphine oxides include, but are not limited to, thetrialkyl phosphine oxide, tri-n-octylphosphine oxide (TOPO), and soforth. Alkyl groups for these phosphine oxides contain about 6-24 carbonatoms per alkyl, and can contain an unsaturated carbon-carbon bond, andeach phosphine oxide has a total number of carbon atoms in all of itsalkyl groups combined of about 10-30 carbon atoms.

Exemplary fatty acids include, but are not limited to, stearic, oleic,palmitic, myristic and lauric acids, as well as other carboxylic acidsof the formula R—COOH, wherein R is a C6-C24 hydrocarbon group and cancontain an unsaturated carbon-carbon bond. It will be appreciated thatthe rate of nanocrystal growth generally increases as the length of thefatty acid chain decreases.

Exemplary ethers and furans include, but are not limited to,tetrahydrofuran and its methylated forms, glymes, and so forth.

Suitable phosphonic and phosphinic acids include, but are not limited tohexylphosphonic acid (HPA), tetradecylphosphonic acid (TDPA), andoctylphosphinic acid (OPA), and are frequently used in combination withan alkyl phosphine oxide such as TOPO. Suitable phosphonic andphosphinic acids are of the formula RPO₃H₂ or R₂PO2H, wherein each R isindependently a C6-C24 hydrocarbon group and can contain an unsaturatedcarbon-carbon bond.

Exemplary pyridines include, but are not limited to, pyridine, alkylatedpyridines, nicotinic acid, and so forth.

Suitable alkenes include, e.g., octadecene and other C4-C24 hydrocarbonswhich are unsaturated.

Nanoparticle core or shell precursors can be represented as a M-sourceand an X-donor. The M-source can be an M-containing salt, such as ahalide, carboxylate, phosphonate, carbonate, hydroxide, or diketonate,or a mixed salt thereof (e.g., a halo carboxylate salt, such asCd(halo)(oleate)), of a metal, M, in which M can be, e.g., Cd, Zn, Mg,Hg, Al, Ga, In, or Tl. In the X-donor, X can be, e.g., 0, S, Se, Te, N,P, As, or Sb. The mixture can include an amine, such as a primary amine(e.g., a C8-C20 alkyl amine). The X donor can include, for example, aphosphine chalcogenide, a bis(trialkylsilyl)chalcogenide, a dioxygenspecies, an ammonium salt, or a tris(trialkylsilyl)phosphine, or thelike.

The M-source and the X donor can be combined by contacting a metal, M,or an M-containing salt, and a reducing agent to form an M-containingprecursor. The reducing agent can include an alkyl phosphine, a 1,2-diolor an aldehyde, such as a C₆-C₂₀ alkyl diol or a C₆-C₂₀ aldehyde.

Suitable M-containing salts include, for example, cadmiumacetylacetonate, cadmium iodide, cadmium bromide, cadmium chloride,cadmium hydroxide, cadmium carbonate, cadmium acetate, cadmium oxide,zinc acetylacetonate, zinc iodide, zinc bromide, zinc chloride, zinchydroxide, zinc carbonate, zinc acetate, zinc oxide, magnesiumacetylacetonate, magnesium iodide, magnesium bromide, magnesiumchloride, magnesium hydroxide, magnesium carbonate, magnesium acetate,magnesium oxide, mercury acetylacetonate, mercury iodide, mercurybromide, mercury chloride, mercury hydroxide, mercury carbonate, mercuryacetate, aluminum acetylacetonate, aluminum iodide, aluminum bromide,aluminum chloride, aluminum hydroxide, aluminum carbonate, aluminumacetate, gallium acetylacetonate, gallium iodide, gallium bromide,gallium chloride, gallium hydroxide, gallium carbonate, gallium acetate,indium acetylacetonate, indium iodide, indium bromide, indium chloride,indium hydroxide, indium carbonate, indium acetate, thalliumacetylacetonate, thallium iodide, thallium bromide, thallium chloride,thallium hydroxide, thallium carbonate, or thallium acetate. SuitableM-containing salts also include, for example, carboxylate salts, such asoleate, stearate, myristate, and palmitate salts, mixed halo carboxylatesalts, such as M(halo)(oleate) salts, as well as phosphonate salts.

Alkyl is a branched or unbranched saturated hydrocarbon group of 1 to100 carbon atoms, preferably 1 to 30 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well ascycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Optionally, an alkyl can contain 1 to 6 linkages selected from the groupconsisting of —O—, —S—, -M- and —NR— where R is hydrogen, or C1-C8 alkylor lower alkenyl.

The X donor is a compound capable of reacting with the M-containing saltto form a material with the general formula MX. The X donor is generallya chalcogenide donor or a phosphine donor, such as a phosphinechalcogenide, a bis(silyl) chalcogenide, dioxygen, an ammonium salt, ora tris(trialkylsilyl) phosphine. Suitable X donors include dioxygen,elemental sulfur, bis(trimethylsilyl) selenide ((TMS)₂Se), trialkylphosphine selenides such as (tri-n-octylphosphine) selenide (TOPSe) or(tri-n-butylphosphine) selenide (TBPSe), trialkyl phosphine telluridessuch as (tri-n-octylphosphine) telluride (TOPTe) orhexapropylphosphorustriamide telluride (HPPTTe),bis(trimethylsilyl)telluride ((TMS)₂Te), sulfur,bis(trimethylsilyl)sulfide ((TMS)₂S), a trialkyl phosphine sulfide suchas (tri-n-octylphosphine) sulfide (TOPS), tris(dimethylamino) arsine, anammonium salt such as an ammonium halide (e.g., NH₄Cl),tris(trimethylsilyl) phosphide ((TMS)₃P), tris(trimethylsilyl) arsenide((TMS)₃As), or tris(trimethylsilyl) antimonide ((TMS)₃Sb). In certainembodiments, the M donor and the X donor can be moieties within the samemolecule.

Ligand Exchange Processes for Coating Nanoparticles

Provided herein are ligand exchange processes that permit efficientconversion of a conventional hydrophobic nanoparticle or populationthereof into a water-dispersible and functionalized nanoparticle orpopulation of nanoparticles. It also permits preparation of smallnanoparticles which are highly stable and bright enough to be useful inbiochemical and biological assays. The resulting nanoparticles can alsobe linked to a target molecule or cell or enzyme (e.g., polymerase) ofinterest.

Typically, the nanoparticle used for this process is a core/shellnanocrystal which is coated with a hydrophobic ligand such astetradecylphosphonic acid (TDPA), trioctylphosphine oxide (TOPO),trioctyl phosphine (TOP), octylphosphonic acid (OPA), and the like, or amixture of such ligands; these hydrophobic ligands typically have atleast one long-chain alkyl group, i.e. an alkyl group having at least 8carbons, or for the phosphine/phosphine oxide ligands, this hydrophobiccharacter may be provided by two or three alkyl chains on a singleligand molecule having a total of at least 10 carbon atoms. Therefore,in some embodiments, the surface of the core/shell nanocrystal orpopulation thereof can be coated with varying quantities of TDPAhydrophobic ligands prior to replacement with hydrophilic ligand(s). Forexample, TDPA can represent at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about80%, at least about 95%, at least about 98%, at least about 99% or moreof the total surface ligands coating the core/shell nanoparticles.Moreover, certain hydrophobic ligands show an unexpected and apparentease of replacement with the hydrophilic ligand. For example,nanoparticles with OPA on the surface have been observed to transferinto aqueous buffer more readily and more completely than the same typeof core-shell with TDPA on the surface. Therefore, in some embodiments,the surface of the core/shell nanocrystal or populations thereof can becoated with varying quantities of OPA hydrophobic ligands prior toreplacement with hydrophilic ligand(s). For example, OPA can representat least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 80%, at least about 95%,at least about 98%, at least about 99% or more of the total surfaceligands coating the core/shell nanocrystal.

In one aspect, provided herein is a “one-step” ligand exchange processto apply various types of ligands to the surface of a nanoparticle, bysubstituting a desired hydrophilic ligand for a conventional hydrophobicligand like TOPO, TOP, TDPA, OPA, and the like. The process steps,comprising: providing a nanocrystal coated with a surface layercomprising a hydrophobic ligand, and dissolved or dispersed in anon-aqueous solvent, contacting the nanocrystal dispersion with a phasetransfer agent and an aqueous solution comprising a hydrophilic ligand,to form a biphasic mixture having an aqueous phase and a non-aqueousphase and maintaining the mixture under conditions that cause thenanocrystal to migrate from the non-aqueous solvent into the aqueousphase. See PCT Application Serial No. PCT/US09/053018 which is expresslyincorporated herein by reference as if set forth in full.

The ‘one-step’ ligand exchange process described herein utilizes phasetransfer catalysts which are particularly effective, and provide fasterexchange reactions. Butanol has been utilized as a phase transfercatalyst for this type of exchange reaction; however, the reaction takesseveral days typically, and requires heating to about 70° C. The timefor this reaction exposes the nanoparticles to these reaction conditionsfor a long period of time, which may contribute to some reduction in itsultimate stability. The embodiments disclosed herein provide moreefficient conditions which achieve ligand exchange more rapidly, thusbetter protecting the nanoparticles. As a result of accelerating theexchange reaction and allowing use of milder conditions, these phasetransfer catalysts produce higher quality nanoparticles.

The phase transfer agent for this process can be a crown ether, a PEG, atrialkylsulfonium, a tetralkylphosphonium, and an alkylammonium salt, ora mixture of these. In some embodiments, the phase transfer agent is18-crown-6, 15-crown-5, or 12-crown-4. In some embodiments, the phasetransfer agent is a PEG, which can have a molecular weight from about500 to about 5000. In some embodiments, the phase transfer agent is atrialkylsulfonium, tetralkylphosphonium, or alkylammonium (includingmonoalkylammonium, dialkylammonium, trialkylammonium andtetralkylammonium) salt.

Tetralkylammonium salts are sometimes preferred as phase transferagents. Examples of suitable tetralkylammonium salts includetriethylbenzyl ammonium, tetrabutylammonium, tetraoctylammonium, andother such quaternary salts. Other tetralkylammonium salts, where eachalkyl group is a C1-C12 alkyl or arylalkyl group, can also be used.Typically, counting all of the carbons on the alkyl groups of atrialkylsulfonium, tetralkylphosphonium, and alkylammonium salt, thephase transfer agent will contain a total of at least 2 carbons, atleast 10 carbons and preferably at least 12 carbon atoms. Each of thetrialkylsulfonium, tetralkylphosphonium, and alkylammonium salts has acounterion associated with it; suitable counterions include halides,preferably chloride or fluoride; sulfate, nitrate, perchlorate, andsulfonates such as mesylate, tosylate, or triflate; mixtures of suchcounterions can also be used. The counterion can also be a buffer orbase, such as borate, hydroxide or carbonate; thus, for example,tetrabutylammonium hydroxide can be used to provide the phase transfercatalyst and a base. Specific phase transfer salts for use in thesemethods include tetrabutylammonium chloride (or bromide) andtetraoctylammonium bromide (or chloride).

Suitable hydrophilic ligands are organic molecules which provide atleast one binding group to associate tightly with the surface of ananocrystal. The hydrophilic ligand typically is an organic moietyhaving a molecular weight between about 100 and 1500, and containsenough polar functional groups to be water soluble. Some examples ofsuitable hydrophilic ligands include small peptide having 2-10 aminoacid residues (preferably including at least one histidine or cysteineresidue), mono- or polydentate thiol containing compounds.

Following ligand exchange, the surface layer can optionally becrosslinked.

In another aspect, provided herein is a “two-step” ligand exchangeprocess to apply various types of ligands to the surface of ananoparticle, by substituting a desired hydrophilic ligand for aconventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and the like.The process involves the removal of phosphonate or phosphinate ligandsfrom the surface of a nanoparticle or nanocrystal by treatment withsulfonate reagents, particularly silylsulfonate derivatives of weakbases or other poorly coordinating groups.

The process steps, comprising: providing a nanocrystal whose surfacecomprises a phosphonate ligand, contacting the nanocrystal with asulfonate reagent in an organic solvent, contacting the sulfonate ligandcoated nanocrystal with a functionalized organic molecule (i.e.,hydrophilic ligand) comprising at least one nanocrystal surfaceattachment group, contacting the nanocrystal dispersion with an aqueoussolution to form a biphasic mixture having an aqueous phase and anon-aqueous phase, and maintaining the biphasic mixture under conditionswhich cause the nanocrystal to migrate from the non-aqueous phase intothe aqueous phase. See PCT Application Serial No. PCT/US09/59456 whichis expressly incorporated herein by reference as if set forth in full.

The result of this removal of phosphonate ligands is replacement of thephosphonates with the weakly coordinating groups. One example is the useof silyl sulfonates, such as trimethylsilyl triflate, to form asulfonate-coated nanoparticle. Triflate is a conventional/common namefor a trifluoromethanesulfonyloxy group, CF₃SO₂O—.

The same type of replacement process can also occur on nanoparticleshaving phosphinic acid ligands of the formula R₂P(═O)—OH or onnanoparticles having carboxylic acid ligands of the formula RC(═O)—OH,which could be incorporated on the surface of a nanocrystal by knownmethods; R can be a C₁-C₂₄ hydrocarbon group in these phosphinates, andthe two R groups can be the same or different. Thus, it is understoodthat when phosphonate-containing nanocrystals are described herein,phosphinate-containing nanocrystals can be used instead, with similarresults.

This process provides a mild and selective method for removingphosphonate, phosphinate, and carboxylate ligands from the surface of ananocrystal. As a result, it provides a way for a user to remove thesegroups and replace them, without removing other ligands which are notdisplaced or affected by the silylsulfonate.

The sulfonate ligands can comprise an alkyl or aryl moiety linked to—SO₃X, where X can represent whatever the sulfonate group is attachedto. For example, where the sulfonate ligand is a sulfonate anion (i.e.,triflate), X would represent a nanocrystal, or the surface of ananocrystal. Some of the sulfonate embodiments disclosed herein can alsobe described with reference to feature ‘A’ of Formula I, as set forthbelow.

wherein R¹, R², R³ and A are each, independently, C1-C10 alkyl or C5-C10aryl; and each alkyl and aryl is optionally substituted.

The alkyl groups for Formula I compounds are independently selected, andcan be straight chain, branched, cyclic, or combinations of these, andoptionally can include a C1-C4 alkoxy group as a substituent. Typically,the alkyl groups are lower alkyls, e.g., C1-C4 alkyl groups which arelinear or branched. Methyl is one suitable example.

The aryl group for the compounds of Formula I can be phenyl, naphthyl ora heteroaryl having up to 10 ring members, and can be monocyclic orbicyclic, and optionally contain up to two heteroatoms selected from N,O and S as ring members in each ring. (It will be understood by thoseskilled in the art that the 5-membered aryl is a heteroaryl ring.)Phenyl is a preferred aryl group; and an aryl group is typically onlypresent if the other organic groups on the silicon other than thesulfonate are lower alkyls, and preferably they are each Me.

Examples of silylsulfonate ligands can include, but are not limited to:(trimethylsilyl)triflate, (triethylsilyl)triflate,(t-butyldimethylsilyl)triflate, (phenyldimethylsily)triflate,trimethylsilyl fluoromethanesulfonate, trimethylsilyl methanesulfonate,trimethylsilyl nitrophenylsulfonate, trimethylsilyltrifluoroethylsulfonate, trimethylsilyl phenylsulfonate, trimethylsilyltoluenesulfonate, diisopropylsilyl bis(trifluoromethanesulfonate),tertbutyldimethylsilyl trifluoromethanesulfonate, triisopropylsilyltrifluoromethanesulfonate and trimethylsilyl chlorosulfonate.

Examples of other sulfonate ligands can include, but are not limited to:trifluoromethanesulfonate (triflate), fluoromethanesulfonate,methanesulfonate (mesylate), nitrophenylsulfonate (nosylate),trifluorethylsulfonate, phenylsulfonate (besylate) and toluenesulfonate(tosylate).

Some suitable examples of the hydrophilic ligand are disclosed, forexample, in Naasani, U.S. Pat. Nos. 6,955,855; 7,198,847; 7,205,048;7,214,428; and 7,368,086. Suitable hydrophilic ligands also includeimidazole containing compounds such as peptides, particularlydipeptides, having at least one histidine residue, and peptides,particularly dipeptides, having at least one cysteine residue. Specificligands of interest for this purpose can include carnosine (whichcontains beta-alanine and histidine); His-Leu; Gly-His; His-Lys;His-Glu; His-Ala; His-His; His-Cys; Cys-His; His-Ile; His-Val; and otherdipeptides where His or Cys is paired with any of the common alpha-aminoacids; and tripeptides, such as Gly-His-Gly, His-Gly-His, and the like.The chiral centers in these amino acids can be the naturalL-configuration, or they can be of the D-configuration or a mixture of Land D. Thus a dipeptide having two chiral centers such as His-Leu can beof the L,L-configuration, or it can be L,D- or D,L; or it can be amixture of diastereomers.

Furthermore, suitable hydrophilic ligands can also include mono- orpolydentate thiol containing compounds, for example: monodentate thiolssuch as mercaptoacetic acid, bidentate thiols such as dihydrolipoic acid(DHLA), tridentate thiols such as compounds of Formula II-VII as shownbelow, and the like.

In compounds of Formula II-VII, R¹, R², R³ can independently be H, halo,hydroxyl, (—(C═O)—C₁-C₂₂, —(C═O)CF₃,) alkanoyl, C₁-C₂₂ alkyl, C₁-C₂₂heteroalkyl, ((CO)OC₁-C₂₂) alkylcarbonato, alkylthio (C₁-C₂₂) or(—(CO)NH(C₁-C₂₀) or —(CO)N(C₁-C₂₀)₂) alkylcarbamoyl. In someembodiments, R¹, R², and R³ are different. In other embodiments, R¹, R²,and R³ are the same.

In compounds of Formula II-VII, IV, and R⁵ can independently be H,C₁-C₂₀ alkyl, C₆-C₁₈ aryl, C₁-C₂₂ heteroalkyl or C₁-C₂₂ heteroaryl. Insome embodiments, R⁴ and R⁵ are different. In other embodiments, and R⁵are the same.

In compounds of Formula II-VII, R⁶ can be H or a polyethylene glycolbased moiety of Formula VIII:

In certain embodiments of Formula VIII, R⁷ can be —NH₂, —N₃, —NHBoc,—NHFmoc, —NHCbz, —COOH, —COOt-Bu, —COOMe, iodoaryl, hydroxyl, alkyne,boronic acid, allylic alcohol carbonate, —NHBiotin, —(CO)NHNHBoc,—(CO)NHNHFmoc or —OMe. In some embodiments, n can be an integer from 1to 100.

In still further embodiments, the tridentate thiol ligands can be acompound of Formula IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII,XIX, XX, XXI, XXII, XXIII or XXIV:

Functionalized TDPA Ligands on Nanoparticles

Provided herein are methods for preparing water-soluble semi-conducting,insulating, or metallic nanoparticles including the steps of admixingone or more nanocrystal precursors and one or more multi-functionalsurface ligands with a solvent to form a solution and heating thesolution to a suitable temperature, and in certain embodiments, methodsmay include the steps of admixing nanocrystal cores, one or morenanocrystal precursors, and one or more multi-functional surface ligandswith a solvent to form a solution and heating the solution to a suitabletemperature. In such embodiments, the one or more multi-functionalsurface ligands may at least include a nanocrystal binding center, alinker, and a functional group, which imparts functionality on thenanocrystal. As used herein the term “functional group” may refer to agroup which affects reactivity, solubility, or both reactivity andsolubility when present on a multi-functional surface ligand.Embodiments can include a wide variety of functional groups which canimpart various types of functionality on the nanocrystal includinghydrophilicity, water-solubility, or dispersibility and/or reactivity,and the functionality may generally not include only hydrophobicity oronly solubility in organic solvents without increasing reactivity. Forexample, a functional group which is generally hydrophobic but whichincreases reactivity such as an alkene or alkyne and certain esters andethers can be encompassed by embodiments, whereas alkyl groups, which donot generally impart reactivity but increase hydrophobicity may beexcluded.

In certain embodiments, the nanoparticles produced by the methods ofsuch embodiments may be coated with ligands which impart watersolubility and/or reactivity on the nanoparticle obviating the need forligand replacement. Without wishing to be bound by theory, eliminatingligand replacement may provide more consistent thermodynamic properties,which may lead to reduction in variability of coating and less loss ofquantum yield, among other improvements in the properties ofnanoparticles produced by the methods embodied herein. Eliminatingligand replacement may also allow for the production of nanoparticleshaving a wide variety of functional groups associated with the coating.In particular, while ligand replacement is generally limited toproduction of nanoparticles having amine and/or carboxylic acidfunctional groups, in various embodiments, the skilled artisan maychoose among numerous functional groups when preparing themulti-functional ligands and may, therefore, generate nanoparticleswhich provide improved water-solubility or water-dispersity and/orsupport improved crosslinking and/or improved reactivity with cargomolecules. See for example PCT Application Serial No. PCT/US09/59117filed Sep. 30, 2009 which are expressly incorporated herein by referenceas if set forth in full.

Solid Surfaces

The methods, compositions, systems and kits disclosed herein can involvethe use of surfaces (e.g., solid surfaces) which can be attachedcovalently or non-covalently with the nanoparticles and/or thebiomolecules (polymerases, nucleotides, target nucleic acid molecules,primers, and/or oligonucleotides) described herein. The attachment canbe reversible or irreversible. The immobilized biomolecules include the:polymerases, nucleotides, target nucleic acid molecules, primermolecules and/or oligonucleotides which are components in the nucleotidebinding and/or nucleotide incorporation reactions. The immobilizednanoparticles and/or biomolecules may be attached to the surface in amanner that they are accessible to components of the nucleotideincorporation reaction and/or in a manner which does not interfere withnucleotide binding or nucleotide incorporation. The immobilizednanoparticles and/or biomolecules may be attached to the surface in amanner which renders them resistant to removal or degradation during theincorporation reactions, including procedures which involve washing,flowing, temperatures or pH changes, and reagent changes. In anotheraspect, the immobilized nanoparticles and/or biomolecules may bereversibly attached to the surface.

The surface may be a solid surface, and includes planar surfaces, aswell as concave, convex, or any combination thereof. The surface maycomprise texture (e.g., etched, cavitated or bumps). The surfaceincludes the inner walls of a capillary, a channel, a well, groove,channel, reservoir, bead, particle, sphere, filter, gel or a nanoscaledevice. The surface can be optically transparent, minimally reflective,minimally absorptive, or exhibit low fluorescence. The surface may benon-porous. The surface may be made from materials such as glass,borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide,plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium,graphite, ceramics, silicon, semiconductor, high refractive indexdielectrics, crystals, gels, polymers, or films (e.g., films of gold,silver, aluminum, or diamond). The surface can include a solid substratehaving a metal film or metal coat.

The immobilized nanoparticles and/or biomolecules may be arranged in arandom or ordered array on a surface. The ordered array includesrectilinear and hexagonal patterns. The distance and organization of theimmobilized molecules may permit distinction of the signals generated bythe different immobilized molecules. The surface can be coated with anadhesive and/or resist layer which can be applied to the surface tocreate the patterned array and can be applied to the surface in anyorder. The adhesive layer can bind/link the nanoparticle or biomolecules(e.g., polymerases, nucleotides, target nucleic acid molecules, primers,and/or oligonucleotides). The resist layer does not bind/link, orexhibits decreased binding/linking, to the nanoparticle or biomolecules(e.g., polymerases, nucleotides, target nucleic acid molecules, primers,and/or oligonucleotides).

The immobilized nucleic acid molecules (e.g., target and/or primermolecules) may be attached to the surface at their 5′ ends or 3′ ends,along their length, or along their length with a 5′ or 3′ portionexposed. The immobilized proteins (e.g., polymerases) can be attached tothe surface in a manner which orients them to mediate their activities(nucleotide binding or nucleotide incorporation).

The surface can be coated to facilitate attachment of nucleic acidmolecules (target and/or primers). For example, a glass surface can becoated with a polyelectrolyte multilayer (PEM) via light-directedattachment (U.S. Pat. Nos. 5,599,695, 5,831,070, and 5,959,837) or viachemical attachment. The PEM chemical attachment can occur by sequentialaddition of polycations and polyanions (Decher, et al., 1992 Thin SolidFilms 210:831-835). In one embodiment, the glass surface can be coatedwith a polyelectrolyte multilayer which terminated with polyanions orpolycations. The polyelectrolyte multilayer can be coated with biotinand an avidin-like compound. Biotinylated molecules (nucleic acidmolecules or polymerases or nanoparticles) can be attached to thePEM/biotin/avidin coated surface (Quake, U.S. Pat. Nos. 6,818,395;6,911,345; and 7,501,245).

Nanoscale Devices

The surface can be the surface of a nanoscale device. The components ofthe nucleotide binding or nucleotide incorporation reaction (e.g.,nanoparticles, polymerase, nucleotides, target nucleic acid molecules,primers and/or oligonucleotides) can be associated with or immobilizedonto the nanoscale device.

The nanoscale device can have microscopic features (e.g., at the micrometer, nano meter size level, or pico meter level) which permitmanipulation or analysis of biological molecules at a nanoscale level.

The nanoscale device can include open or enclosed (i.e., sealed)structures (e.g., nanostructures) including: channels, slits, pores,wells, pillars, loops, arrays, pumps valves. The nanostructures can havelength, width, and height dimensions. The nanostructures can be linearor branched, or can have inlet and/or outlet ports. The branchednanostructures (e.g., branched channels) can form a T or Y junction, orother shape and geometries.

The nanostructure dimensions can be between about 10-25 nm, or about25-50 nm, or about 50-100 nm, or about 100-200 nm, or about 200-500 nm,or about 500-700 nm, or about 700-900 nm, or about 900-1000 nm. Thenanostructures can have a trench width equal to or less than about 150nanometers. The nanostructures can be wells which are 50-10,000 nm indiameter. The nanostructures can have a trench depth equal to or lessthan about 200 nanometers (e.g., 50-100 nm thickness).

The nanoscale device can comprise one or a plurality of nanostructures,typically more than 5, 10, 50, 100, 500, 1000, 10,000 and 100,000nanostructures for binding, holding, streaming, flowing, washing,flushing, or stretching samples. The samples can include thenanoparticles, polymerase, nucleotides, target nucleic acid molecules,primers and/or oligonucleotides. The fluid which runs through thenanoscale device can be liquid, gas or slurry. Nanoscale devices arealso known as nanofluidic devices.

Nanoscale devices and/or their component nanostructures may befabricated from any suitable substrate including: silicon, carbon,glass, polymer (e.g., poly-dimethylsiloxane), metals, boron nitrides,nickel, platinum, copper, tungsten, titanium, aluminum, chromium, gold,synthetic vesicles, carbon nanotubes, or any combination thereof.

The nanoscale devices and and/or nanostructures may be fabricated usingany suitable method, including: lithography; photolithography;diffraction gradient lithography (DGL); nanoimprint lithography (NIL);interference lithography; self-assembled copolymer pattern transfer;spin coating; electron beam lithography; focused ion beam milling;plasma-enhanced chemical vapor deposition; electron beam evaporation;sputter deposition; bulk or surface micromachining; replicationtechniques such as embossing, printing, casting and injection molding;etching including nuclear track or chemical etching, reactiveion-etching, wet-etching; sacrificial layer etching; wafer bonding;channel sealing; and combinations thereof.

The nanoscale device can be used to react, confine, elongate, mix, sort,separate, flow, deliver, flush, wash, or enrich the nanoparticles orbiomolecules, or the intermediates or products of nucleotideincorporation. For example, the target nucleic acid molecule (e.g.,nucleic acid molecules, or chromosomal or genomic DNA) can be elongatedusing pulsed field electrophoresis, or in a nanofluidic device via flowstretching (with or without tethering) or confinement elongation.Elongated nucleic acid molecules can be used to: measure the contourlength of a nucleic acid molecule, locate landmark restriction sitesalong the length of the molecule, or detect sequencing reactions alongthe molecule (Schwartz, U.S. Pat. Nos. 6,221,592, 6,294,136 and U.S.Published App. Nos. 2006/0275806 and 2007/0161028). In one aspect, thenanostructure can be one or more nanochannels, which are capable oftransporting a macromolecule (e.g., nucleic acid molecule) across itsentire length in elongated form. In another aspect, the nanostructurecan detect an elongated macromolecule, or detect sequencing of a singlenucleic acid molecule.

The nanochannels can be enclosed by surmounting them with a sealingmaterial using suitable methods. See, for example, U.S. Publication No.2004/0197843. The nanoscale device can comprise a sample reservoircapable of releasing a fluid, and a waste reservoir capable of receivinga fluid, wherein both reservoirs are in fluid communication with thenanofluidic area. The nanoscale device may comprise a microfluidic arealocated adjacent to the nanofluidic area, and a gradient interfacebetween the microfluidic and nanofluidic area which reduces the localentropic barrier to nanochannel entry. See, for example, U.S. Pat. No.7,217,562.

The nanoscale device comprising a nanochannel array can be used toisolate individual nucleic acid molecules prior to sequencing, whereinthe sample population of nucleic acid molecules is elongated anddisplayed in a spatially addressable format. Isolation of the nucleicacid molecules to be sequenced may be achieved using any suitablenanoscale device which comprises nanostructures or nanofluidicconstrictions of a size suited to achieve isolation and separation ofthe test nucleic acid molecule from other sample components in a mannerwhich will support direct sequencing of the test molecule in situ. Forexample, a nucleic acid molecule, such as a chromosome, is isolated froma sample mixture using a nanofluidic device which is capable ofreceiving a sample comprising mixed population of nucleic acid moleculesand elongating and displaying them in an ordered format without the needfor prior treatment or chemical attachment to a support.

The nanoscale device supports analysis of intact chromosomes without theneed for fragmentation or immobilization of sequencing components. Thenanoscale device comprises at least one nanostructure, typically ananochannel, which is designed to admit only a single polymeric moleculeand elongate it as it flows through the nanostructure. Suitablenanoscale devices have been described, for example, in U.S. Pat. No.6,635,163 (nanofluidic entropic trapping and sieving devices). Suitablenanoscale devices comprise microfluidic and nanofluidic areas separatedby a gradient interface which reduces the local entropic barrier tonanochannel entry thereby reducing clogging of the device at themicrofluidic-nanofluidic interface. See, for example, Cao, U.S. Pat. No.7,217,562 and U.S. Pub. No. 2007/0020772.

The nanoscale device can include an array of nanochannels. Introductionof a sample comprising a mixed population of nucleic acid molecules intothe nanoscale device results in the isolation and elongation of a singlenucleic acid molecule within each nanostructure, so that an entirepopulation of nucleic acid molecules is displayed in an elongated andspatially addressable format. After the nucleic acid molecules enter andflow through their respective nanochannel, they are contacted with oneor more components of a nucleotide incorporation reaction mixture, andthe progress of the incorporation reaction is monitored using suitabledetection methods. The ordered and spatially addressable arrangement ofthe population allows signals to be detected and monitored along thelength of each nucleic acid molecule. Separate sequencing reactionsoccur within each nanochannel. The spatially addressable nature of thearrayed population permits discrimination of signals generated byseparate priming events, and permitting simultaneous detection andanalysis of multiple priming events at multiple points in the array. Theemission data can be gathered and analyzed to determine thetime-sequence of incorporation events for each individual nucleic acid(DNA) in the nanochannel array. Nanoscale devices can permit thesimultaneous observation of macromolecules in multiple channels, therebyincreasing the amount of sequence information obtainable from a singleexperiment and decreasing the cost of sequencing of an entire genome.See, for example, U.S. Pub. No. 2004/0197843, also U.S. Ser. Nos.61/077,090, filed on Jun. 30, 2008, and 61/089,497, filed on Aug. 15,2008, and 61/090,346, filed on Aug. 20, 2008.

In one embodiment, the nanoscale device can include a flow cell whichincludes a two-sided multi-channel flow cell comprising multipleindependently-addressable sample channels and removable loading blocksfor sample loading (Lawson, U.S. published patent application No.2008/0219888).

In another embodiment, the nanoscale device can include a light sourcefor directing light to the nucleotide incorporation reaction, a detector(e.g., photon detector), a camera, and/or various plumbing componentssuch as microvalves, micropumps, connecting channels, andmicroreservoirs for controlled flow (in and/or out) of the reagents ofthe nucleotide incorporation reactions. The reagents can be pulledthrough the inlet or outlet ports via capillary action, or by vacuum(Lawson, U.S. published patent application No. 2008/0219890; and Harris,et al., 2008 Science 320:106-109, and Supplemental Materials and Methodsfrom the supporting online material), or moved via a pressure-drivenfluidics system. The reagents can be pulled through the inlet or outletports using a passive vacuum source (Ulmer, U.S. Pat. No. 7,276,720).

In another embodiment, the nucleotide incorporation methods can bepracticed in a nanoscale device such as a patterned metal masked arraywhich includes a metal layer disposed on a glass support, where themetal layer is perforated with holes ranging in size from 50-10,000 nm.The holes can be any shape including round, rectilinear, triangular,slit, and the like. The metal layer can have a thickness of about 50-100nm. The metal layer can be gold, chrome, silver, aluminum, titanium,nickel, platinum, copper, tungsten, titanium-tungsten, carbon, carbonnanotubes, nanoparticles, or polymers. The surface can be spin-coatedwith an imaging resist using e-beam or photo resist procedures. Themetal can be global-coated using evaporation or sputtering procedures.The exposure step can be achieved using e-beam or photomask lithography.See for example, U.S. Ser. No. 61/245,248, filed Sep. 23, 2009.

The nanoparticles and biomolecules (e.g., polymerases, nucleotides,target nucleic acid molecules, primers, and/or oligonucleotides) can beisolated, modified, sorted, collected, distributed, linked and/orimmobilized using suitable procedures and devices.

The nanoparticles and biomolecules (e.g., polymerases, nucleotides,target nucleic acid molecules, primers, and/or oligonucleotides) can beused presently for any procedure described herein, or can be stored orpreserved for later use by employing suitable procedures.

Modified Surfaces

The surface can be chemically or enzymatically modified to have one ormore reactive groups, including amines, aldehyde, hydroxyl, sulfate orcarboxylate groups, which can be used to attach the surface to thenanoparticles, polymerases, nucleotides, target nucleic acid molecules,primers, and/or oligonucleotides.

Attaching Nucleic Acid Molecules to the Surface

Nucleic acid molecules can be attached to a surface. The target nucleicacid molecules, primers, and/or oligonucleotides can be modified attheir 5′ or 3′ end, or internally, to carry a reactive group which canbind to a reactive group on the surface. Typically, the surface istreated or untreated to provide reactive groups such as silanol,carboxyl, amino, epoxide, and methacryl groups. The nucleic acidmolecules can be treated or untreated to provide reactive groupsincluding: amino, hydroxyl, thiol, and disulfide. The nucleic acidmolecules can include non-natural nucleotides having reactive groupwhich will attach to a surface reactive group. For example, thenon-natural nucleotides include peptide nucleic acids, locked nucleicacids, oligonucleotide N3′→P5′ phosphoramidates, andoligo-2′-O-alkylribonucleotides.

In one aspect, nucleic acid molecules modified with one or more aminogroups at the 5′ or 3′ end, or internally, can be attached to modifiedsurfaces.

In another aspect, the nucleic acid molecules can be attached at their5′ ends with one or more amino groups, including: a simple amino group;a short or long tethering arm having one or more terminal amino groups;or amino-modified thymidine or cytosine. The tethering arms can belinear or branched, have various lengths, charged or uncharged,hydrophobic, flexible, cleavable, or have one or multiple terminal aminogroups. The number of plural valent atoms in a tethering arm may be, forexample, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30 or a larger numberup to 40 or more.

In another aspect, the 3′ end of nucleic acid molecules can be modifiedto carry an amino group. Typically, the amino group is initiallyprotected by a fluorenylmethylcarbamoyl (Fmoc) group. To expose theamino group, the protecting group can be removed and acylated with anappropriate succinimidyl ester, such as an N-hydroxy succinimidyl ester(NHS ester).

In another aspect, the nucleic acid molecules can carry internal aminogroups for binding to the solid surface. For example, 2′ amino modifiednucleic acid molecules can be produce by methoxyoxalamido (MOX) orsuccinyl (SUC) chemistry to produce nucleotides having amino linkersattached at the 2′ C of the sugar moiety.

In another aspect, the surface can be modified to bind the aminomodified nucleic acid molecules. For example, 5′ amino-modified nucleicacid molecules can be attached to surfaces modified with silane, such asepoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res.22:2121-2125; W. G. Beattie et al., 1995 Mol. Biotechnol. 4:213-225) orisothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465).Acylating reagents can be used to modify the surface for attaching theamino-modified nucleic acid molecules. The acylating reagents include:isothiocyanates, succinimidyl ester, and sulfonyl chloride. Theamino-modified nucleic acid molecules can attach to surface amino groupswhich have been converted to amino reactive phenylisothiocyanate groupsby treating the surface with p-phenylene 1,4 diisothiocyanate (PDC). Inother methods, the surface amino groups can be reacted withhomobifunctional crosslinking agents, such as disuccinimidylcaronate(DCS), disuccinimidyloxalate (DSO), phenylenediisothiocyanate (PDITC) ordimethylsuberimidate (DMS) for attachment to the amino-modified nucleicacid molecules. In another example, metal and metal oxide surfaces canbe modified with an alkoxysilane, such as 3-aminopropyltriethoxysilane(APTES) or glycidoxypropyltrimethoxysilane (GOPMS).

In another aspect, succinylated nucleic acid molecules can be attachedto aminophenyl- or aminopropyl-modified surfaces (B. Joos et al., 1997Anal. Biochem. 247: 96-101).

In yet another aspect, a thiol group can be placed at the 5′ or 3′ endof the nucleic acid molecules. The thiol group can form reversible orirreversible disulfide bonds with the surface. The thiol attached to the5′ or 3′ end of the nucleic acid molecule can be a phosphoramidate. Thephosphoramidate can be attached to the 5′ end usingS-trityl-6-mercaptohexyl derivatives.

In another aspect, the thiol-modified nucleic acid molecules can beattached to a surface using heterobifunctional reagents (e.g. crosslinkers). For example, the surface can be treated with an alkylatingagent such as iodoacetamide or maleimide for linking with thiol modifiednucleic acid molecules. In another example, silane-treated surfaces(e.g., glass) can be attached with thiol-modified nucleic acid moleculesusing succinimidyl 4-(malemidophenyl)butyrate (SMPB).

In another aspect, the nucleic acid molecule can be modified to carrydisulfide groups can be attached to thiol-modified surfaces (Y. H.Rogers et al., 1999 Anal. Biochem. 266:23-30).

Still other aspects include methods which employ modifying reagents suchas: carbodiimides (e.g., dicyclohexylcarbodiimide, DCC),carbonyldiimidazoles (e.g., carbonyldiimidazole, CDIz), and potassiumperiodate. The nucleic acid molecules can have protectivephotoprotective caps (Fodor, U.S. Pat. No. 5,510,270) capped with aphotoremovable protective group. DMT-protected nucleic acid moleculescan be immobilized to the surface via a carboxyl bond to the 3′ hydroxylof the nucleoside moiety (Pease, U.S. Pat. No. 5,599,695; Pease et al.,1994 Proc. Natl. Acad. Sci. USA 91(11):5022-5026). The nucleic acidmolecules can be functionalized at their 5′ ends with activated1-O-mimethoxytrityl hexyl disulfide 1′-[(2-cyanoethyl)-N,N-diisopropyl)]phosphoramidate (Rogers et al., 1999 Anal. Biochem. 266:23). Exemplarymethods of attaching nucleic acid molecules to suitable substrates aredisclosed, for example, in Schwartz, U.S. Pat. Nos. 6,221,592, 6,294,136and U.S. Published App. Nos. 2006/0275806 and 2007/0161028 (Schwartz etal.). Linking agents, can be symmetrical bifunctional reagents, such asbis succinimide (e.g., bis-N-hydroxy succinimide) and maleimide(bis-N-hydroxy maleimide) esters, or toluene diisocyanate can be used.Heterobifunctional cross-linkers include: m-maleimido benzoyl-N-hydroxysuccinimidyl ester (MBS); succinimidyl-4-(p-maleimido phenyl)-Butyrate(SMPB); and succinimidyl-4-(N-Maleimidomethyl)Cyclohexane-1-Carboxylate(SMCC) (L. A. Chrisey et al., 1996 Nucleic Acids Res. 24:3031-3039). Inone example, a glass surface can be layered with a gold (e.g., about 2nm layer) which is reacted with mercaptohexanoic acid. Themercaptohexanoic acid can be placed in a patterned array. Themercaptohexanoic acid can be reacted with PEG. The PEG can be reacted tobind nucleic acid molecules such as the target nucleic acid molecules.

In another aspect, the target nucleic acid molecule can be linked to anamine-functionalized solid surface. In one embodiment, theamine-functionalized solid surface can be a spot surrounded by PEGmolecules, where the target molecule preferentially binds theamine-functionalized spots (see Fry, et al., U.S. Ser. No. 61/245,248,filed on Sep. 23, 2009).

Capture Probes:

The surfaces, nanoparticles, polymerases, nucleotides, target nucleicacid molecules, primers, and/or oligonucleotides can be attached to eachother in any combination via capture nucleic acid probes.

For example, the surface may comprise capture nucleic acid probes whichform complexes with single or double stranded nucleic acid molecules. Inone embodiment, the capture probes anneal with target nucleic acidmolecules. The capture probes include oligonucleotide clamps (U.S. Pat.No. 5,473,060). The parameters for selecting the length and sequence ofthe capture probes are well known (Wetmur 1991 Critical Reviews inBiochemistry and Molecular Biology, 26: 227-259; Britten and Davidson,chapter 1 in: Nucleic Acid Hybridization: A Practical Approach, Hames etal, editors, IRL Press, Oxford, 1985). The length and sequence of thecapture probes may be selected for sufficiently stability during lowand/or high stringency wash steps. The length of the capture probesranges from about 6 to 50 nucleotides, or from about 10 to 24nucleotides, or longer.

Attaching Proteins to the Solid Surface

In one aspect, the surface can be modified to attach the proteinmolecules (e.g., polymerases) via covalent or non-covalent linkage. Thepolymerases may be attached to the surface via covalent cross-linkingbridges, including disulfide, glycol, azo, sulfone, ester, or amidebridges. Some exemplary methods for attaching polymerases to a surfaceare disclosed in U.S. Pat. Nos. 7,056,661, 6,982,146, 7,270,951,6,960,437, 6,255,083, 7,229,799 and published application U.S. No.2005/0042633.

The polymerases can be modified at their amino- or carboxyl-terminalends, or internally, to carry a reactive group which can bind to areactive group on the surface.

The polymerases can be attached to the modified surfaces using standardchemistries including: amination, carboxylation or hydroxylation. Theattachment agents can be cyanogen bromide, succinimide, aldehydes, tosylchloride, photo-crosslinkable agents, epoxides, carbodiimides orglutaraldehyde (in: Protein immobilization: Fundamentals andApplications, Richard F. Taylor, ed. (M. Dekker, New York, 1991). Thesurface can be treated or untreated to provide reactive groups such assilanol, carboxyl, amino, epoxide, and methacryl groups. The proteinmolecules can be treated or untreated to provide reactive groupsincluding: amino, hydroxyl, thiol, and disulfide. The surface can becoated with an electron-sensitive compound such as polymethylmethacrylate-like material (PMMA).

The polymerases can be attached to a surface which is untreated ormodified via physical or chemical interaction. See Nakanishi for areview of protein immobilization methods (K. Nakanishi, 2008 CurrentProteomics 5:161-175).

The polymerases can be adsorbed onto a surface. The adsorption can occurvia ion exchange, charge-charge interaction, or hydrogen bondinteractions. The adsorption can occur on to untreated surfaces,including polystyrene, polyvinylidene fluoride (PVDF), glass coated withpoly-lysine (H. Ge 2000 Nucl. Acids Res. 28: e3; B. B. Haab, et al.,2001 Genome Biol. 2: R4-13; Zhu and Snyder 2003 Curr. Opin. Chem. Biol.7: 55-63), or onto surfaces having hydrophobic properties (Y. Sanghak,et al., 2006 Curr. Appl. Phys. 6: 267-70).

The polymerases can be attached to the surface using a hydrogel (P.Arenkov, et al., 2000 Anal. Biochem. 278: 123-31; S. Kiyonaka, et al.,2004 Nat. Mater. 3: 58-64).

The polymerases can be linked to an affinity His-tag (e.g., 6×His-tag(SEQ ID NO: 63)) which interacts with Ni²⁺, Co²⁺, or Cu²⁺ surfaces (T.Nakaji-Hirabayashi, et al., 2007 Biomaterials 28: 3517-29; R.Vallina-Garcia, et al., 2007 Biosens. Bioelectron. 23: 210-7; T. Cha, etal., 2004 Proteomics 4: 1965-76; T. Cha, et al., 2005 Proteomics 5:416-9). For example, the polymerases can be a fusion protein whichincludes the His-tag sequence. The glass surface can be functionalizedwith a chelate group by treating with nitrotriacetic acid (NTA) orimidoacetic acid (IDA) and reacted with Ni²⁺ or Cu²⁺, respectively.

The polymerases can be attached to the surface via chemisorption betweena thiol (e.g., SH group of cysteines) on the polymerase and a goldsurface (S. V. Rao, et al., 1998 Mikrochim. Acta 128: 127-43).

The polymerases can be attached to the surface via a Schiff's baselinkage reaction. For example, a glass surface can be silanized withsilane, polysilane, trimethoxysilane, or aminosilane. The silanizedglass surface can interact with amino groups (e.g., lysine) on thepolymerase (MacBeath and Schreiber 2000 Science 289: 1760-1763; H. Zhu,et al., 2000 Nat. Genet. 26: 283-289). Metal and metal oxide surfacescan be modified with an alkoxysilane, such as3-aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane(GOPMS).

The polymerases can be immobilized via protein coil-coil interactionbetween a heterodimeric Leu zipper pair (J. R. Moll, et al., 2001Protein Sci. 10: 649-55; K. Zhang, et al., 2005 J. Am. Chem. Soc. 127:10136-7). For example, the surface can be functionalized to bind one ofthe zipper proteins, and the polymerases can be linked with the otherzipper protein. The polymerases can be fusion proteins which include azipper protein sequence. The glass surface can be coated with abifunctional silane coupling reagent comprising aldehyde (e.g.,octyltrichlorosilane (OTC)) and functionalized with a hydrophobicelastin mimetic domain (ELF) as a hydrophobic surface anchor whichserves to bind a leucine zipper sequence. The anchored zipper sequencecan interact with a partner leucine zipper sequence linked to thepolymerases.

The polymerases can be immobilized via an acyl transfer reaction. Forexample, transglutaminase (TGase) can catalyze an acyl transfer reactionbetween a primary amino group and a carboxyamide group (J. Tominaga, etal., 2004 Enz. Microb. Technol. 35: 613-618). In one embodiment,carboxyamide groups from a casein-coated surface can react with theprimary amine groups (e.g., lysine as a peptide tag or part of thepolymerase) on the polymerases. In another embodiment, the amine groupson the surface can react with carboxyamide groups (e.g., glutamine-tagor glutamine groups on the polymerases).

The polymerases can be immobilized via interaction between an affinitypeptide sequence (e.g., motif) and its cognate peptide binding partner.For example, the affinity motif could bind a protein kinase. In oneembodiment, the affinity motif comprises the “minimal” motif,R-X-X-S*/T* (T. R. Soderling 1996 Biochim. Biophys. Acta 1297: 131-138),including peptide motifs RRATSNVFA (SEQ ID NO:17), RKASGPPV (SEQ IDNO:18), or LRRASLG (SEQ ID NO:19), which bind a calmodulin-dependentprotein kinase.

Oriented poly-His tagged protein molecules can be immobilized on to aglass surface modified with PEG and reacted with a chelate group such asiminodiacetic acid (IDA) or nitrolotriacetic acid (NTA), and metal ionssuch as Ni²⁺ or Cu²⁺ (T. Cha, et al., 2004 Proteomics 4:1965-1976).

EDAC chemistry can be use to link a carboxylated silica surface to anavidin. The avidin can bind to a biotinylated protein (e.g.,polymerase). The avidin-silica surface can bind one or more biotinylatedprotein molecules, or bind more than one type of biotinylated protein(e.g., binds biotinylated polymerase).

In one aspect, a peptide linker can be used to attach the proteinmolecules (e.g., polymerases) to the nanoparticle or to the solidsurface. The peptide linkers can be part of a fusion protein comprisingthe amino acid sequences of the polymerases. The fusion protein caninclude the peptide linker positioned at the N- or C-terminal end or inthe interior of the fusion protein. In another embodiment, the peptidelinkers can be separate linkers which are attached to the protein andthe solid surface or nanoparticle.

For example, the peptide linker can be a flexible linker comprising theamino acid sequence GGGGSGGGGSAAAGSAA (SEQ ID NO:20). In anotherexample, the peptide linker can be a rigid linker comprising the aminoacid sequence GAAAKGAAAKGSAA (SEQ ID NO:21). In another example, thepeptide linker can be a poly-lysine linker, comprising between about4-15 lysine residues (e.g., 12 lysine residues). BS3 coupling(bis(sulfo-succinimidyl)suburate) can be used to attach the poly-lysinelinkers to PEG-amine groups on the solid surfaces or on nanoparticles.In yet another example, the peptide linker can be a poly-cysteine linkercomprising between about 4-15 cysteine residues (e.g., 12 cysteineresidues). SMCC coupling(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) can beused to attach the poly-cysteine linkers to PEG-amine groups on solidsurfaces or on nanoparticles. In yet another example, the peptide linkercan be a transglutaminase tag comprising the amino acid sequence PKPQQF(SEQ ID NO:22) or PKPQQFM (SEQ ID NO:23). The transglutaminase tag canprovide site specific attachment of the protein (polymerase) to thesolid surface or nanoparticle. Transglutaminase enzyme can catalyze anacyl transfer reaction between the γ-carboxyamide group of an acceptorglutamine residue and a primary amine donor on the solid surface ornanoparticles. In yet another example, the peptide linker can be aprotein kinases (PKA) tag comprising the amino acid sequence LRRASL (SEQID NO: 62). The PKA tag can provide site specific attachment of theprotein (polymerase) to the solid surface or nanoparticle. SPDP(N-succinimidyl 3-(2-pyridyldithio) propionate) and iodoacetic acid areheterobifunctional cross-linking agents which can react with amines andsulfhydryl groups to link proteins to the solid surfaces ornanoparticles.

In yet another embodiment, the peptide linker can include apoly-histidine tag:

(SEQ ID NO: 14) MNHLVHHHHHHIEGRHMELGTLEGS, or (SEQ ID NO: 15)MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGS, or (SEQ ID NO: 16)MHHHHHHLLGGGGSGGGGSAAAGSAAR.

In one embodiment, the solid surface can be modified to provide avidin(or avidin-like) binding groups. In one embodiment, the surface materialis glass. In another embodiment, the glass surface is reacted withsilane or its derivative. In another embodiment, the glass surface isreacted with PEG, biotin, and avidin (or avidin-like protein) to provideavidin (or avidin-like) binding sites. In yet another embodiment, theglass surface is reacted with PEG and avidin (or avidin-like protein) toprovide avidin (or avidin-like) binding site. The binding sites on theglass slide can attach to the nanoparticles, proteins (e.g.,polymerases, or any fusion proteins thereof), target nucleic acidmolecules, primers, or oligonucleotides.

In another embodiment, the polymerase (or polymerase fusion protein) islinked to the surface. In another embodiment, the solid surface can bemodified for binding to a His-tagged protein. In another embodiment, thepolymerase can be a biotinylated protein bound to a surface which iscoated with avidin or avidin-like protein. In another embodiment, thepolymerase can be a poly-His-tagged protein bound to a nickel-conjugatedsurface. In another embodiment, the polymerase (or polymerase fusionprotein) can be linked to a nanoparticle. In another embodiment,polymerase and nanoparticle can be separately linked to the surface. Theimmobilized polymerase can bind the target nucleic acid molecule, whichmay or may not be base-paired with the polymerization initiation. Theimmobilized polymerase can bind the nucleotide and/or can incorporatethe nucleotide onto the polymerization initiation site.

Reducing Non-Specific Binding

In one aspect, the surfaces, nanoparticles, polymerases, nucleotides,target nucleic acid molecules, primers, and/or oligonucleotides can bemodified to reduce non-specific binding by dyes or nucleotides. Forexample, the surface can be coated with sugar molecules (e.g., mono ordisaccharides as described in Jogikalmath, U.S. 2008/0213910), silane(Menchen, U.S. Ser. No. 11/943,851), and/or PEG to reduce non-specificbinding with dyes and/or nucleotides. Silane includes:N-(3-aminopropyl)3-mercapto-benzamide; 3-aminopropyl-trimethoxysilane;3-mercaptopropyl-trimethoxysilane; 3-(trimethoxysilyl)propyl-maleimide;and 3-(trimethoxysilyl)propyl-hydrazide. In another example, thenanoparticles can be reacted with bovine serum albumin (BSA) to reducenon-specific binding to polymerases.

Linking Methods

In some embodiments, the surfaces, reporter moieties (including, e.g.,energy transfer moieties, nanoparticles and organic dyes), polymerases,nucleotides and nucleic acid molecules (including, e.g., targets,primers and/or oligonucleotides) can be linked to each other, in anycombination and in any order, using well known linking chemistries. Suchlinkage can optionally include a covalent bond and/or a non-covalentbond selected from the group consisting of an ionic bond, a hydrogenbond, an affinity bond, a dipole-dipole bond, a van der Waals bond, anda hydrophobic bond.

In some embodiments, the linking procedure used to link thebiomolecules, reporter moieties and/or surfaces of the presentdisclosure comprises a chemical reaction that includes formation of oneor more covalent bonds between a first and second moiety, resulting inthe linkage of the first moiety to the second moiety. In someembodiments, the chemical reaction occurs between a first group of themoiety and a second group of the second moiety. Such chemical reactioncan include, for example, reaction of activated esters, acyl azides,acyl halides, acyl nitriles, or carboxylic acids with amines or anilinesto form carboxamide bonds. Reaction of acrylamides, alkyl halides, alkylsulfonates, aziridines, haloacetamides, or maleimides with thiols toform thioether bonds. Reaction of acyl halides, acyl nitriles,anhydrides, or carboxylic acids with alcohols or phenols to form anester bond. Reaction of an aldehyde with an amine or aniline to form animine bond. Reaction of an aldehyde or ketone with a hydrazine to form ahydrazone bond. Reaction of an aldehyde or ketone with a hydroxylamineto form an oxime bond. Reaction of an alkyl halide with an amine oraniline to form an alkyl amine bond. Reaction of alkyl halides, alkylsulfonates, diazoalkanes, or epoxides with carboxylic acids to form anester bond. Reaction of an alkyl halides or alkyl sulfonates with analcohol or phenol to form an ether bond. Reaction of an anhydride withan amine or aniline to form a carboxamide or imide bond. Reaction of anaryl halide with a thiol to form a thiophenol bond. Reaction of an arylhalide with an amine to form an aryl amine bond. Reaction of a boronatewith a glycol to form a boronate ester bond. Reaction of a carboxylicacid with a hydrazine to form a hydrazide bond. Reaction of acarbodiimide with a carboxylic acid to form an N-acylurea or anhydridebond. Reaction of an epoxide with a thiol to form a thioether bond.Reaction of a haloplatinate with an amino or heterocyclic group to forma platinum complex. Reaction of a halotriazine with an amine or anilineto form an aminotriazine bond. Reaction of a halotriazines with analcohol or phenol to form a triazinyl ether bond. Reaction of an imidoester with an amine or aniline to form an amidine bond. Reaction of anisocyanate with an amine or aniline to form a urea. Reaction of anisocyanate with an alcohol or phenol to form a urethane bond. Reactionof an isothiocyanate with an amine or aniline to form a thiourea bond.Reaction of a phosphoramidate with an alcohol to form a phosphite esterbond. Reaction of a silyl halide with an alcohol to form a silyl etherbond. Reaction of a sulfonate ester with an amine or aniline to form analkyl amine bond. Reaction of a sulfonyl halide with an amine or anilineto form a sulfonamide bond. Reaction of a thioester with thiol group ofa cysteine followed by rearrangement to form an amide bond. Reaction ofan azide with an alkyne to form a 1,2,3-traizole.

In some embodiments, water-insoluble substances can be chemicallymodified in an aprotic solvent such as dimethylformamide,dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform.Similar modification of water-soluble substances can be accomplishedusing reactive compounds to make them more readily soluble in organicsolvents.

Linkage to Surface

In some embodiments the biomolecules and/or reporter moieties of thepresent disclosure are linked to a surface. Optionally, such linkage canresult in reversible or non-reversible immobilization of thenanoparticles, polymerases, nucleotides, nucleic acid molecules,primers, and/or oligonucleotides onto the surface. Non-limiting examplesof such linkage can include: nucleic acid hybridization, proteinaptamer-target binding, non-specific adsorption, and solventevaporation. In some embodiments, the biomolecule that is linked to asurface is a polymerase (such as, for example, a polymerase fusionprotein). The polymerase can be attached to a surface via a linkercomprising an anchor or tethering moiety. The anchor or tethering moietycan be flexible or rigid. The anchor or tether can orient thepolymerase, or polymerase fusion protein, in a manner that does notinterfere with the nucleotide binding and/or polymerase activity.

Conjugation Methods—Biomolecules

Linkage of biomolecules to reporter moieties, surfaces and/or to eachother can be accomplished by any suitable method (for example, Brinkleyet al., 1992 Bioconjugate Chem. 3: 2). In some embodiments, abiomolecule can comprise a single type of reactive site (as is typicalfor polysaccharides), or it can comprise multiple types of reactivesites, e.g., amines, thiols, alcohols, phenols, may be available (as istypical for proteins). Conjugation selectivity can be obtained byselecting an appropriate reactive moiety. For example, modification ofthiols with a thiol-selective reagent such as a haloacetamide ormaleimide, or modification of amines with an amine-reactive reagent suchas 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (variously known asEDC or EDAC), an activated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

In some embodiments, the biomolecule can be linked to the reportermoiety through a bond selected from group consisting of: a covalentbond, a hydrogen bond, a hydrophilic bond, a hydrophobic bond, anelectrostatic bond, a Van der Waals bond, and an affinity bond.

In some embodiments, the biomolecule comprises a peptide and the bond isa covalent bond formed between an amine group of a lysine residue of thebiomolecule and an amine-reactive moiety, wherein the amine reactivemoiety is linked to the reporter moiety. In some embodiments, thebiomolecule comprises a peptide and the bond is a covalent bond formedbetween a carboxy group of an amino acid residue of the biomolecule anda maleimide moiety, wherein the maleimide moiety is linked to thereporter moiety.

In some embodiments, the biomolecule can be linked to a reporter moiety,such as, for example a nanoparticle. Optionally, the nanoparticlefurther comprises at least one carboxyl group on its surface, and theone or more biomolecules or fragments at least one primary amine group,and the cross-linking agent EDC is employed to form a covalent amidebond between the at least one nanoparticle and the one or morebiomolecules or fragments.

In some embodiments, the biomolecule can be attached to a reportermoiety (including, e.g., a FRET donor or acceptor moiety) using anysuitable chemical linking procedure, including chemical linkingprocedures that are known in the art. In some embodiments, the at leastone biomolecule or biologically active fragment can be operably linkedto the nanoparticle via chemical linking procedures. Many linkingprocedures are well known in the art, including: maleimide, iodoacetyl,or pyridyl disulfide chemistry which targets thiol groups onpolypeptides; or succinimidyl esters (NHS), sulfonyl chlorides,iso(thio)cyanates, or carbonyl azide chemistry which targets primaryamines in a polypeptide, and dichlorotriazine-based linking procedures.Additional exemplary linking procedures are described in more detailherein.

In some embodiments, the appropriate reactive compounds can be dissolvedin a nonhydroxylic solvent (usually DMSO or DMF) in an amount sufficientto give a suitable degree of conjugation when added to a solution of theprotein to be conjugated. These methods have been used to prepareprotein conjugates from antibodies, antibody fragments, avidins,lectins, enzymes, proteins A and G, cellular proteins, albumins,histones, growth factors, hormones, and other proteins. The resultingprotein (e.g., polymerase) attached to the energy transfer or reportermoiety can be used directly or enriched, e.g., chromatographicallyenriched to separate the desired linked compound from the undesiredunlinked compound. Several linking procedures are described in U.S. Pat.No. 5,188,934. Other suitable linking procedures are also known in theart.

When conjugating biomolecules to nanoparticles, the residual, unreactedcompound or a compound hydrolysis product can be removed by dialysis,chromatography or precipitation. The presence of residual, unconjugatedmoieties can be detected by methods such as thin layer chromatographywhich elutes the unconjugated forms away from its conjugate. In someembodiments, the reagents are kept concentrated to obtain adequate ratesof conjugation.

Modification to Facilitate Linkage

In some embodiments, the surfaces, reporter moieties (including, e.g.,dyes and/or nanoparticles) and/or biomolecules (including, e.g.,polymerases, nucleotides and nucleic acid molecules) disclosed hereincan be modified to facilitate their linkage to each other. Suchmodification can optionally include chemical or enzymatic modification.The modification can be practiced in any combination and in any order.In some embodiments, the modification can mediate covalent ornon-covalent linkage of the surfaces, reporter moieties and/orbiomolecules with each other.

In some embodiments, the biomolecule can be attached, fused or otherwiseassociated with a moiety that facilitates purification and/or isolationof the biomolecule. For example, the moiety can be an enzymaticrecognition site, an epitope or an affinity tag that facilitatespurification of the biomolecule.

In some embodiments, the polymerase can include an amino acid analogwhich provides a reactive group for linking to the nanoparticle, target,substrate and/or surface. For example, the amino acid analog can beproduced using a cell (e.g., bacterial cell) which is geneticallyengineered to have a 21 amino acid genetic code which is capable ofinserting the amino acid analog into the encoded polymerase (or fusionprotein). The inserted amino acid analog can be used in a linkingchemistry procedure to attach the polymerase (or fusion protein) to theenergy transfer donor moiety, biomolecule or the surface.

His Tag Modification

In some embodiments, the biomolecule is a protein and is modified with aHis tag. In some embodiments, the His tag may be fused directly with theprotein; alternatively, a linker comprising various lengths of aminoacid residues can be placed between the protein and the His tag. Thelinker can be flexible or rigid.

Optionally, the presence of the His tag can facilitate purification ofthe protein. For example, His tagged protein can be purified from a rawbacterial lysate by contacting the lysate with any suitable affinitymedium comprising bound metal ions to which the histidine residues ofthe His-tag can bind, typically via chelation. The bound metal ions cancomprise, e.g., zinc, nickel or cobalt, to which the His tag can bindwith micromolar affinity. Suitable affinity media include Ni Sepharose,NTA-agarose, HisPur® resin (Thermo Scientific, Pierce Protein Products,Rockford, Ill.), or Talon® resin (Clontech, Mountain View, Calif.). Theaffinity matrix can then be washed with suitable buffers, e.g.,phosphate buffers, to remove proteins that do not specifically interactwith the cobalt or nickel ion. Washing efficiency can be improved by theaddition of 20 mM imidazole. The biomolecule can optionally be elutedfrom the proteins are usually eluted with 150-300 mM imidazole). Thepurity and amount of purified biomolecule can then be assessed usingsuitable methods, e.g., SDS-PAGE and Western blotting.

Optionally, the His tag can be fused to a suitable amino acid sequencethat facilitates removal of the His-tag using a suitable endopeptidase.Alternatively, the His tag may be removed using a suitable exopeptidase,for example the Qiagen TAGZyme exopeptidase.

In some embodiments, the His tag can facilitate linkage of thebiomolecule to a metal surface, for example, a surface comprising Zn²⁺,Ni²⁺, Co²⁺, or Cu²⁺ ions. Optionally, the His-tag can facilitate linkageof the biomolecule to the surface of a nanoparticle comprising one ormore metal ions, typically via chelation interactions, as described inmore detail herein.

Linkers

Suitable linkers can be used to link the biomolecules (including, e.g.,the polymerases, nucleotides and nucleic acid molecules), the labels(including, e.g., nanoparticles, organic dyes, energy transfer moietiesand/or other reporter moieties) and/or the surfaces of the presentdisclosure to each other, in any combination. The linkers can beattached (to the surfaces, nanoparticles, polymerases, nucleotides,target nucleic acid molecules, primers, oligonucleotides, reportermoieties, and/or energy transfer moieties) via covalent bonding,non-covalent bonding, ionic bonding, hydrophobic interactions or anycombination thereof. The type and length of the linker can be selectedto optimize tethering, proximity, flexibility, rigidity, or orientation.The attachment can be reversible or non-reversible.

Suitable linkers include without limitation homobifunctional linkers andheterobifunctional linkers. For example, heterobifunctional linkerscontain one end having a first reactive functionality to specificallylink to a first molecule, and an opposite end having a second reactivefunctionality to specifically link to a second molecule. Depending onsuch factors as the molecules to be linked and the conditions in whichthe method of strand synthesis is performed, the linker can vary inlength and composition for optimizing properties such as stability,length, FRET efficiency, resistance to certain chemicals and/ortemperature parameters, and be of sufficient stereo-selectivity or sizeto link a nanoparticle to the biomolecule such that the resultantconjugate is useful reporting biomolecular activity such as approach,bonding, fusion or catalysis of a particular chemical reaction. Linkerscan be employed using standard chemical techniques and include but notlimited to, amine linkers for attaching reporter moieties to nucleotides(see, for example, U.S. Pat. No. 5,151,507); a linker containing aprimary or secondary amine for linking a reporter moiety to anucleotide; and a rigid hydrocarbon arm added to a nucleotide base (see,for example, Science 282:1020-21, 1998).

In some embodiments, the linker comprises a polyethylene glycol (PEG) orPEG derivative. See, e.g., U.S. Provisional Applications 61/086,750;61/102,709; 61/102,683; and 61/102,666. Such PEG moieties can befunctionalized at one or both ends. In some embodiments,functionalization at both ends with the same reactive moiety can beemployed to create a homobifunctional PEG derivative. Some examples ofhomobifunctional PEG derivatives include without limitationCOOH-PEG-COOH; NH2-PEG-NH2; and MAL-PEG-MAL (where MAL denotes amaleimide group).

The linker moiety can optionally include: a covalent or non-covalentbond; amino acid tag; chemical compound (e.g., polyethylene glycol);protein-protein binding pair (e.g., biotin-avidin); affinity coupling;capture probes; or any combination of these.

Optionally, the linker can be selected such that they do notsignificantly interfere with the function or activity of thebiomolecules, reporter moieties and/or surfaces that it links to eachother. For example, when the biomolecule is a polymerase, the linker canbe selected such that it does not significantly interfere withnucleotide binding to the polymerase, or with cleavage of thephosphodiester bonds, or with nucleotide incorporation, or with releaseof the polyphosphate product, or with translocation of the polymerase orwith energy transfer, or with emission of a detectable signal.

In some embodiments, the linker can comprise a single covalent bond or aseries of covalent bonds. Optionally, the linker can be linear,branched, bifunctional, trifunctional, homofunctional, orheterofunctional. The linker can be cleavable. The linkers can be rigidor flexible. The linker can be capable of energy transfer. The linkercan be a chemical chain or a chemical compound. The linker can beresistant to heat, salts, acids, bases, light and chemicals. The linkercan include a short or long spacer, a hydrophilic spacer, or an extendedspacer.

In another embodiment, the rigid linker can be used to improve a FRETsignal. Examples of rigid linkers include benzyl linkers, proline orpoly-proline linkers (S. Flemer, et al., 2008 Journal Org. Chem.73:7593-7602), bis-azide linkers (M. P. L. Werts, et al., 2003Macromolecules 36:7004-7013), and rigid linkers synthesized by modifyingthe so-called “click” chemistry scheme which is described by Megiattoand Schuster 2008 Journal of the Am. Chem. Soc. 130:12872-12873. In yetanother embodiment, the linker can be an energy transfer linkersynthesized using methods described in U.S. published patent applicationNo. 2006/0057565, which is incorporated in its entirety. In yet anotherembodiment, the spacer linking moiety can be a cationic arginine spaceror an imidazolium spacer molecule.

In some embodiments, the linker moiety comprises about 1-40 pluralvalent atoms or more selected from the group consisting of C, N, O, Sand P. The number of plural valent atoms in a linker may be, forexample, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, or 40, or more. Alinker may be linear or non-linear; some linkers have pendant sidechains or pendant functional groups (or both). Examples of such pendantmoieties are hydrophilicity modifiers, for example solubilizing groupslike, e.g., sulfo (—SO₃H— or —SO³—). In some embodiments, a linker iscomposed of any combination of single, double, triple or aromaticcarbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,carbon-oxygen bonds and carbon-sulfur bonds. Exemplary linking membersinclude a moiety which includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, andthe like. Linkers may by way of example consist of a combination ofmoieties selected from alkyl, alkylene, aryl, —C(O)NH—, —C(O)O—, —NH—,—S—, —O—, —C(O)—, —S(O)_(n)— where n is 0, 1, 2, 3, 4, 5, or 6-memberedmonocyclic rings and optional pendant functional groups, for examplesulfo, hydroxy and carboxy.

In some embodiments, the linker can result from “click” chemistriesschemes (see, e.g., Gheorghe, et al., 2008 Organic Letters 10:4171-4174)which can be used to attach any combination of biomolecules, reportermoieties and surfaces as disclosed herein to each other

In one aspect, the linker can attach two or more energy transfer orreporter moieties to each other (the same type or different types ofmoieties). In another aspect, a trifunctional linker (e.g., Graham, U.S.published patent application No. 2006/0003383) can be linked to twofluorescent dye moieties (the same type or different types) to amplifythe fluorescent signal upon nucleotide binding or nucleotideincorporation. For example, a trifunctional linker can be linked to twoenergy transfer acceptor moieties, or to an energy transfer acceptor anda reporter moiety. In another example, multiple trifunctional linkerscan be linked to each other, which can be linked to multiple fluorescentdyes for dendritic amplification of the fluorescent signal (e.g.,Graham, U.S. published patent application No. 2007/0009980).

In some embodiments, the linker can be a cleavable linker such as, forexample, a photocleavable linker, a chemically cleavable linker or aself-cleaving linker.

In some embodiments, the linker is a self-cleaving linker. Optionally,such linker can be a trimethyl lock or a quinone methide linker, whichcan each optionally link to two energy transfer acceptor and/or reportermoieties and the nucleotide.

In some embodiments, the linkers can be cleavable where cleavage ismediated by a chemical reaction, enzymatic activity, heat, acid, base,or light. For example, photo-cleavable linkers include nitrobenzylderivatives, phenacyl groups, and benzoin esters. Many cleavable groupsare known in the art and are commercially available. See, for example,J. W. Walker, et al., 1997 Bioorg. Med. Chem. Lett. 7:1243-1248; R. S.Givens, et al., 1997 Journal of the American Chemical Society119:8369-8370; R. S. Givens, et al., 1997 Journal of the AmericanChemical Society 119:2453-2463; Jung et al., 1983 Biochem. Biophys.Acta, 761: 152-162; Joshi et al., 1990 J. Biol. Chem., 265: 14518-14525;Zarling et al., 1980 J. Immunol., 124: 913-920; Bouizar et al., 1986Eur. J. Biochem., 155: 141-147; Park et al., 1986 J. Biol. Chem., 261:205-210; and Browning et al., 1989 J. Immunol., 143: 1859-1867; see alsoU.S. Pat. No. 7,033,764. A broad range of cleavable, bifunctional (bothhomo- and hetero-bifunctional) spacer arms with varying lengths arecommercially available.

A rigid linker can be used. In some embodiments, use of a rigid linkercan be useful in improving a FRET signal. Examples of rigid linkersinclude benzyl linkers, proline or poly-proline linkers (S. Flemer, etal., 2008 Journal Org. Chem. 73:7593-7602), bis-azide linkers (M. P. L.Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkerssynthesized by modifying the so-called “click” chemistry scheme that isdescribed by Megiatto and Schuster 2008 Journal of the Am. Chem. Soc.130:12872-12873.

In yet another embodiment, the linker can be an energy transfer linkersynthesized using methods described in U.S. Published Patent ApplicationNo. 2006/0057565.

In yet another embodiment, the linker can comprise a spacer, for examplea cationic arginine spacer or an imidazolium spacer molecule.

In some embodiments, the linker can be a fragmentable linker, includingnon-lamellar “detergent-like” micelles or lamellar vesicle-like micellessuch as small unilamellar vesicles or liposomes (“SUVs”), smallmultilamellar vesicles or liposomes (SMVs”), large unilamellar vesiclesor liposomes (“LUVs”) and/or large multilamellar vesicles or liposomes(“LMVs”) (see U.S. application Ser. No. 11/147,827) and see U.S.Application Nos. 60/577,995, and Ser. No. 12/188,165.

In some embodiments, the linker can include multiple amino acid residues(e.g., arginine) which serve as an intervening linker between theterminal phosphate group and the reporter moiety. For example, thelinker can be can four arginine residues which connect a dye moiety to anucleotide comprising one or more phosphate groups, wherein the linkerlinks the dye moiety to the terminal phosphate group of the nucleotide.

In some embodiments, linkers can be used to attach energy transfer orreporter moieties to nucleotides using any suitable linking procedure,including: amine linkers for attaching reporter moieties to nucleotides(see, for example, Hobbs, U.S. Pat. No. 5,151,507); a linker comprisinga primary or secondary amine for operably linking a reporter moiety to anucleotide; and a rigid hydrocarbon arm added to a nucleotide base (see,for example, R. F. Service, 1998 Science 282(5391):1020-21). Someexemplary linking procedures for attaching energy transfer or reportersmoieties to base molecules are provided in European Patent Application87310256.0; International Application PCT/US90/05565; Marshall, 1975Histochemical Journal 7:299-303; and Barone et al., 2001 Nucleosides,Nucleotides, and Nucleic Acids, 20(4-7): 1141-1145. Other examplesinclude linkers for attaching energy transfer or reporter moieties tooligonucleotides synthesized using phosphoramidate to incorporateamino-modified dT (see Mathies, U.S. Pat. No. 5,707,804).

PEG Linkers

In one aspect, a linker comprising a polymer of ethylene oxide can beused to attach the surfaces, reporter moieties (including, e.g., dyesand nanoparticles), polymerases, nucleotides and/or nucleic acidmolecules of the present disclosure to each other in any combination.Non-limiting examples of such polymers of ethylene oxide includepolyethylene glycol (PEG), including short to very long PEG, branchedPEG, amino-PEG-acids, PEG-amines, PEG-hydrazines, PEG-guanidines,PEG-azides, biotin-PEG, PEG-thiols, and PEG-maleinimides. For example,PEG includes: PEG-1000, PEG-2000, PEG-12-OMe, PEG-8-OH, PEG-12-COOH, andPEG-12-NH₂. In some embodiments, the PEG molecule may be linear orbranched. In some embodiments, it can have a molecular weight greaterthan or approximately equal to 1000, 2000, 3000, 4000, 5000 or greater.

In some embodiments, functionalization with different reactive moietiescan be used create a heterobifunctional PEG derivative comprisingdifferent reactive groups at each end. Such heterobifunctional PEGs canbe useful in linking two entities, where a hydrophilic, flexible andbiocompatible spacer is needed. Some examples of heterobifunctional PEGderivatives include without limitation Hydroxyl PEG Carboxyl(HO-PEG-COOH): Thiol PEG Carboxyl (HS-PEG-COOH); Hydroxyl PEG Amine(HO-PEG-NH2); t-Boc Amine PEG Amine (TBOC-PEG-NH2); Amine PEG Carboxyl(NH2-PEG-COOH); t-Boc Amine PEG NHS Ester (TBOC-PEG-NHS); FMOC Amine PEGNHS Ester (FMOC-PEG-NHS): Acrylate PEG NHS Ester (ACLT-PEG-NHS);Maleimide PEG Carboxyl (MAL-PEG-COOH); Maleimide PEG Amine(MAL-PEG-NH2), including the TFA Salt thereof; Maleimide PEG NHS Ester(MAL-PEG-NHS); Biotin PEG NHS Ester (BIOTIN-PEG-NHS); BiotinPolyethylene Glycol Maleimide (BIOTIN-PEG-MAL); OPSS PEG NHS Ester(OPSS-PEG-NHS).

Optionally, the PEG derivative can be a multi-arm PEG derivative. Insome embodiments, the multi-arm PEG derivative can be a PEG derivativehaving a core structure comprising pentaerythritol (including, forexample, 4arm PEG Amine (4ARM-PEG-NH2); 4arm PEG Carboxyl(4ARM-PEG-COOH); 4arm PEG Maleimide (4ARM-PEG-MAL); 4arm PEGSuccinimidyl Succinate (4ARM-PEG-SS); 4arm PEG Succinimidyl Glutarate(4ARM-PEG-SG)); a PEG derivative having a core structure comprisinghexaglycerin (including, for example, 8arm PEG Amine (8ARM-PEG-NH2);8arm PEG Carboxyl (8ARM-PEG-COOH); 8arm PEG Succinimidyl Succinate(8ARM-PEG-SS); 8arm PEG Amine (8ARM-PEG-SG); PEG derivative having acore structure comprising tripentaerythritol (including, for example,8arm PEG Amine (8ARM(TP)-PEG-NH2); 8arm PEG Carboxyl(8ARM(TP)-PEG-COOH); 8arm PEG Succinimidyl Succinate (8ARM(TP)-PEG-SS);8arm PEG Amine (8ARM(TP)-PEG-SG)). Optionally, end groups forheterobifunctional PEGs can include maleimide, vinyl sulfones, pyridyldisulfide, amine, carboxylic acids and NHS esters. The activated PEGderivatives can then be used to attach the PEG to the desiredbiomolecule and/or nanoparticle. Optionally, one or both ends of the PEGderivative can be attached to the N-terminal amino group or theC-terminal carboxylic acid of a protein-comprising biomolecule.

Signal Detection

The methods, compositions, systems and kits disclosed herein can involvethe use of a detection system for optical or spectral detection of asignal, or a change in a signal, generated (emitted) by the energytransfer moiety(ies) or reporter moiety(ies) in the nucleotide bindingor nucleotide incorporation reactions.

The systems and methods can detect and/or measure a signal, or a changeor an amount of change of an optical or spectral characteristic of asignal (e.g., fluorescence or quenching) from a reporter moiety, such asan energy transfer donor and/or acceptor moiety. The change in thesignal can include changes in the: intensity of the signal; duration ofthe signal; wavelength of the signal; amplitude of the signal; durationbetween the signals; and/or rate of the change in intensity, duration,wavelength or amplitude. The change in the signal can include a changein the ratio of the change of the energy transfer donor relative tochange of the energy transfer acceptor signals.

The detection system comprises: excitation illumination, opticaltransmission elements, detectors, and/or computers.

In one aspect, detecting radiation emitted by an excited energy transferor reporter moiety during nucleotide binding comprises: the nucleotide,which can be labeled with a FRET acceptor, binds the polymerase whichcan be labeled with a FRET donor, bringing the FRET acceptor/donor pairin proximity to each other, and the FRET donor can be excited resultingin energy transfer to the FRET acceptor which emits a signal which isdetectable by the detection system.

The detection system comprises excitation illumination which can excitethe energy transfer or reporter moieties which produce a detectablesignal. The excitation illumination can be electromagnetic energy, suchas radio waves, infrared, visible light, ultraviolet light, X-rays orgamma rays. The source of the electromagnetic radiation can be a laser,which possesses properties of mono-chromaticity, directionality,coherence, polarization, and/or intensity. The laser can produce acontinuous output beam (e.g., continuous wave laser) or produce pulsesof light (e.g., Q-switching or mode-locking). The laser can be used in aone-photon or multi-photon excitation mode. The laser can produce afocused laser beam. The wavelength of the excitation electromagneticradiation can be between about 325-850 nm, or between about 325-752 nm,or between about 330-752 nm, or between about 405-752 nm. The laser canbe generated by a mercury, xenon, halogen, or other lamps.

The wavelength and/or power of the excitation illumination can beselected to avoid interfering with or damaging the polymerase enzymaticactivities. The excitation illumination can be focused on a stationaryposition or moved to a different field of view (FOV). The excitationillumination can be directed at a nucleotide incorporation reactionwhich is: in a liquid volume (e.g., aqueous or oil); on a surface; in oron a nanodevice; in a waveguide; or in an evanescent illumination system(e.g., total internal reflection illumination). The excitationillumination can pass through a transparent or partially transparentsurface which is conjugated (covalently or non-covalently) with thecomponents of the nucleotide incorporation reaction.

The energy transfer moiety (e.g., a FRET donor) can be excited by theexcitation illumination at a particular wavelength, and transmit theexcitation energy to an acceptor moiety which is excited and emits asignal at a longer wavelength. The energy transfer moiety or reportermoiety can undergo multi-photon excitation with a longer wavelength,typically using a pulsed laser.

The detection system comprises suitable optical transmission elementswhich are capable of transmitting light from one location to anotherwith the desired refractive indices and geometries. The opticaltransmission elements transmit the excitation illumination and/or theemitted energy in an unaltered or altered form. The optical transmissionelements include: lens, optical fibers, polarization filters (e.g.,dichroic filters), diffraction gratings (e.g., etched diffractiongrating), arrayed waveguide gratings (AWG), optical switches, mirrors,dichroic mirrors, dichroic beam splitter, lenses (e.g., microlens andnanolens), collimators, filters, prisms, optical attenuators, wavelengthfilters (low-pass, band-pass, or high-pass), wave-plates, and delaylines, or any combination thereof.

The detection system comprises suitable detectors which are capable ofdetecting and/or distinguishing the excitation illumination and/or theemitted energy. A wide variety of detectors are available in the art,including: single or multiple channel detectors, high-efficiency photondetection systems, optical readers, charge couple devices (CCD),photodiodes (e.g. avalanche photo diodes (APD)), APD arrays, cameras,electron-multiplying charge-coupled device (EMCCD), intensified chargecoupled device (ICCD), photomultiplier tubes (PMT), multi-anode PMT,complementary metal oxide semiconductor (CMOS) chip(s), and a confocalmicroscope equipped with any of the foregoing detectors. The location ofthe nucleotide incorporation reaction can be aligned, with respect tothe excitation illumination and/or detectors, to facilitate properoptical transmission.

Suitable detection methods can be used for detecting and/ordistinguishing the excitation illumination (or change in excitationillumination) and/or the emitted energy (or change in emitted energy),including: confocal laser scanning microscopy, Total Internal Reflection(TIR), Total Internal Reflection Fluorescence (TIRF), near-fieldscanning microscopy, far-field confocal microscopy, wide-fieldepi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, multi-foci multi-photon, or any combinationsthereof.

The signals emitted from different energy transfer moieties can beresolved using suitable discrimination methods which are based on:fluorescence resonance energy transfer measurements; photoconversion;fluorescent lifetime measurements; polarization; fluorescent lifetimedetermination; correlation/anti-correlation analysis; Raman; intensity;ratiometric; time-resolved methods; anisotropy; near-field or far fieldmicroscopy; fluorescence recovery after photobleaching (FRAP); spectralwavelength discrimination; measurement and separation of fluorescencelifetimes; fluorophore identification; background suppression, parallelmulti-color imaging, or any combination thereof. See, for example, J. R.Lakowitz 2006, in: “Principles of Fluorescence Spectroscopy”, ThirdEdition. If the different nucleotides are labeled with different energytransfer or reporter moieties, then resolving the emitted signals can beused to distinguish between the different nucleotides which bind thepolymerase and/or which are incorporated by the polymerase.

In one embodiment, a system and method for detecting radiation emittedby an excited energy transfer or reporter moiety comprises: anillumination source (e.g., a laser) which produces the excitation energy(e.g., one or multi-photon excitation radiation) which is directed, viaa dichroic beam splitter, through a lens, and through a transparentsurface or onto a surface, where the nucleotide binding reaction or thenucleotide incorporation reaction is attached to the surface or is in asolution. The excitation illumination excites the energy transfer orreporter moiety (e.g., fluorescent dye and/or nanoparticle) resulting inemitted radiation (or a change in radiation) which passes back throughthe dichroic beam splitter and is directed to the detector (or an arrayof detectors) which is capable of identifying and/or resolving the typeof emission. Information about the detected emitted signals is directedto the computer where the information is registered and/or stored. Thecomputer can process the registered and/or stored information todetermine the identity of the nucleotide which bound the polymerase orthe identity of the incorporated nucleotide.

In one aspect, the system and method for detecting radiation emitted byan excited energy transfer or reporter moiety includes amultifluorescence imaging system. For example, the different nucleotidesmay each be linked to different FRET acceptor moieties. The FRETacceptor moieties can be selected to have minimal overlap between theabsorption and emission spectra, and the absorption and emission maxima.The multifluorescence imaging system can simultaneously (orsubstantially simultaneously) detect signals from the FRET acceptormoieties, and resolve the signals. Such multifluorescent imaging can beaccomplished using suitable filters, including: band pass filters, imagesplitting prisms, band cutoff filters, wavelength dispersion prisms,dichroic mirrors, or diffraction gratings, or any combination thereof.

In another aspect, the multifluorescence imaging system is capable ofdetecting the signals emitted by the different energy transfer andreporter moieties attached to the different nucleotides. Such a systemcan include special filter combinations for each excitation line and/oreach emission band. In one embodiment, the detection system includestunable excitation and/or tunable emission fluorescence imaging. Fortunable excitation, light from a light source can pass through a tuningsection and condenser prior to irradiating the sample. For tunableemissions, emissions from the sample can be imaged onto a detector afterpassing through imaging optics and a tuning section. The tuning sectionscan be controlled to improve performance of the system.

In yet another aspect, the detection system comprises an optical trainwhich directs signals emitted from an organized array onto differentlocations of an array-based detector to detect multiple optical signalsfrom multiple locations. The optical trains typically include opticalgratings and/or wedge prisms to simultaneously direct and separatesignals having differing spectral characteristics from differentaddressable locations in an array to different locations on anarray-based detector, e.g., a CCD.

In another aspect, the detection methods include detecting photon burstsfrom the labeled nucleotides during incorporation. The photon bursts canbe the fluorescent signals emitted by the energy transfer moiety whichis linked to the nucleotide. The photon bursts can be a FRET event. Themethods can additionally include analyzing the time trace of the photonbursts. The methods can be practiced using time-resolved fluorescencecorrelation spectroscopy.

Nucleotide incorporation reactions using nucleotides labeled at theterminal phosphate with a fluorescent dye have been previouslydemonstrated (Sood, U.S. published patent application No. 2004/0152119;and Kumar, U.S. Pat. No. 7,393,640). Furthermore, fluorescence detectionof single molecule nucleotide incorporation reactions has been routinelyobtained (Kao, U.S. Pat. No. 6,399,335; and Fuller, U.S. Pat. No.7,264,934).

The nucleotide labeling strategy can be used as a basis for selectingany suitable detection system for detecting and/or resolving signalsemitted by the nucleotide binding reaction or the nucleotideincorporation reaction. Exemplary labeling and detection strategiesinclude but are not limited to optical train and TIRF detection methodssuch as those disclosed in U.S. Pat. No. 6,423,551; and U.S. Pub. Nos.2006/0176479, 2007/0109536, 2007/0111350, and 2007/0250274.

Sequence Analysis of Detected Signals

Following detection of the sample emissions, the raw emission data canbe analyzed to identify events involving nucleotide polymerization. Insome embodiments, the emissions can be analyzed in single moleculeformat to identify nucleotide polymerization.

In one aspect, a labeled enzyme conjugate is a labeled polymeraseconjugate, and a time series of nucleotide incorporations by the labeledpolymerase conjugate is detected and analyzed to deduce the orderedsequence of nucleotides (identifying the nucleotide bases) in the singlenucleic acid substrate that is being replicated by the polymerase.

In one exemplary embodiment, the labeled polymerase conjugate comprisesan energy transfer moiety that undergoes FRET with the energy transfermoiety of an incoming labeled nucleotide that is polymerized by thepolymerase of the conjugate. Nucleic acid sequence analysis is performedby first analyzing the raw emission data to computationally determinethe occurrence of a FRET event. In some embodiments, FRET events i.e., adetectable change in a signal produced from a donor or acceptorresulting from a change in the distance between the donor and acceptor,can be identified using a Hidden Markov Model (HMM)-based or equivalentgeneralized likelihood ratio test that determines the location of anintensity change point based on individual photon arrival times; thistest can then be applied recursively to an entire single moleculeintensity trajectory, thus finding each change points. The true numberof states accessible to the system is then computed. See, e.g., Watkinset al., “Detection of Intensity Change Points in Time-ResolvedSingle-Molecule Measurements” J. Phys. Chem. B., 109(1):617-628 (2005).An exemplary FRET detection method using this technique is describedherein in Example 14.

In one aspect, a system can collect and analyze chemical and/or physicalevent data occurring at one or a plurality of locations within a viewingvolume or field of an imaging apparatus. In some embodiments, the systemcomprises a sample subsystem for containing a sample to be detected andanalyzed, where the sample includes at least one moiety (e.g., enzyme,substrate, reporter moiety, etc) having detectable property thatundergoes a change before, during or after one or a sequence of chemicaland/or physical events involving the moiety. The system can alsoincludes a detection apparatus having a viewing field that permits thedetection of changes in the detectable property of the moiety within theviewing field. The system also includes a data processing subsystemconnected to the imaging apparatus for collecting, storing and analyzingdata corresponding to the chemical and/or physical events occurring atdefinable locations in the viewing field involving one or more moietieswithin the viewing field of the imaging subsystem. The data processingsubsystem converts the data into classifications of events according theevent type determined by a set of parameters defining or characterizingeach event type. See, e.g., U.S. Published Patent Application No.2007/0250274, Volkov et al. which is incorporated herein as if set forthin full.

In one aspect, FRET events can be identified by computationallydetermining the occurrence of an anti-correlated FRET event (typicallyinvolving a correlated decrease in donor signal and increase in acceptorsignal). In one exemplary embodiment, FRET events corresponding tointeractions between a donor fluorophore associated with a first moiety,e.g., a polymerase and an acceptor fluorophore associated with a secondmoiety, e.g., a nucleotide can be analyzed by first collecting orreceiving data from a viewing volume of an imaging apparatus such as anCCD or iCCD detection system. In some embodiments, the data can be in asingle data channel or a plurality of data channels, each data channelrepresenting a different frequency range of emitted fluorescent light,e.g., one channel can include fluorescent light data emitted by a donor,a donor channel, while other channels include fluorescent light dataemitted by an acceptor, an acceptor channel, or by another donor, asecond donor channel. In certain embodiments, a channel will exit foreach different fluorophore being detected simultaneously. In someembodiments, the acceptors are selected so that they can be separatelyidentified based on detectable attributes of their signals e.g.,intensity, frequency shifts, signal duration, attenuation, etc. Afterdata collection, the separate data channels are spatially correlatedwithin the viewing volume so that active fluorophores can be spatiallyand temporally related, called calibration or registration. The goal ofcalibration is to determine the pixel coordinates in each quadrant thatcorrespond to a single position on the slide or a single location withinthe viewing field—to make sure that the data in each channel isspatially coincident over the viewing field and through time ofdetection. After reading the configuration file and the open log file,calibrations, if any, are loaded from the command line. After loadingthe calibration information, a corresponding directory is read asspecified in the command line with all subdirectories, for each one.This read step includes: (1) scanning for calibration stacks, and ifthere are some not matched by the available calibrations, generate newcalibrations out of them; (2) scanning for stacks; if there are some,assume this directory is a slide; and (3) scanning the directory pathfor a date and slide name comprising reaction conditions such as donoridentity, acceptor identity, buffers, etc. See, for example, U.S.Published Patent Application No. 2007/0250274, Volkov et al.

Once FRET events have been identified, they can be analyzed to determinethe order and sequence of nucleotide incorporations.

Analysis of Fluorescence Data to Extrapolate Sequence Information

To convert the observed fluorescence emissions detected during thesequencing reaction into nucleotide sequence information, the raw datacomprising a movie of observed emissions was first processed by using aHidden Markov Model (HMM)-based algorithm or equivalent to detect andidentify FRET events. The subsequent detected FRET events were filteredand filtered sequences were aligned. Each of these two steps, FRET eventdetection and sequence analysis, are described in more detail below.

Detection of FRET Events

The analysis underlying FRET event detection is designed to processspatially correlated movie(s) comprising sequence fluorescence emissiondata, and extract time-series of interest from those data. A movietypically contains one or more channels where each channel representsthe same spatial location at different wavelengths. The analysis chainbegins with the submission of one or more movies to the analysis machinevia a comprehensive user interface. The user interface requires the userto input various parameters that describe the movie(s) (e.g. channelregions, dye emission properties, etc.). Once this data is submitted themovie(s) are then processed by the image analysis software where asliding window of N frames propagates through the movie calculating atemporal local average of the frames within the window. At each positionof the window in the movie, the local average image is then furtherprocessed and enhanced using well known image processing algorithms anda record of the maximum projection of all the local average images isrecorded to produce a global image of the movie. This global image isthe input into a spot identification algorithm which produces a set ofspots identified by a unique spot id, its x and y location and itscorresponding channel, for the sake convenience referred to as aspot-tuple. Each set of spots for a given channel is then registered tothe set of spots in every other channel. In this way a set of spottuples is constructed. If a detected spot in one channel does not have acorresponding detected spot in another channel, then the position of theundetected spot using the transformation between the two channels andthe location of the detected spot is inferred. Once a complete set ofspot tuples is constructed the movie is iterated over and at each framethe amplitude of each spot is calculated and appended to the appropriatetime-series.

The collection of time-series from a spot tuple consists of time-seriesfrom donor and corresponding acceptor channels. This collection iscalled a Vector Time-Series (VTS). The FRET detection process startswith a data segmentation step using a Markov Chain Monte-Carlo (MCMC)algorithm. Each segment of VTS is modeled by a multivariate Gaussianmodel, with each of the channel modeled by a mean and a standarddeviation. This model establishes a baseline for each channel, fromwhich quantities such as “Donor Down” and “Acceptor Up” can becalculated. A Hidden Markov Model (HMM) or equivalent algorithm is usedto model the observed data. The underlying states consist of a nullstate, a blink state and a number of FRET states (one for each acceptorchannel). Each state has its emission probability, which reflects thestate's corresponding physical concept. FRET states are characterized bysignificant “donor down” and “acceptor up” signals. Blink state ischaracterized by significant “donor down” with no “acceptor up”. Nullstate is characterized by no “donor down” and no “acceptor up”. Giventhe observed VTS signal, the emission matrix, and a state transitionprobability matrix, the most probable state path can be computed usingthe Viterbi algorithm. This state path assigns each of the frames to astate. Temporally neighboring FRET frames are grouped into FRET events.For each of the detected FRET events, a list of event features arecalculated, including event duration, signal average, signal to noiseratio, FRET efficiency, probability of event, color calling and otherfeatures. This list of events and corresponding features are stored in afile.

The final stage of the automated analysis generates a report summarizingthe results in the form of a web page containing summary image,statistics of the spots and FRET detection, together with line intensityplots and base call plots.

Using the above process, the movie data obtained from the sequencingreactions was analyzed to detect and identify FRET events according tothe process described above. The FRET events were then processed toidentify sequences as described below.

Sequence Analysis

The string of FRET events from the same spot-tuple are then aligned to areference sequence. Each color call in the string is associated with anucleotide, creating a DNA sequence. That DNA sequence and a referencesequence are fed into a Smith-Waterman alignment or equivalent algorithmto determine where the read comes from in the template sequence and thesimilarity between the sequences.

Kits

Provided herein are kits for conducting the nucleotide binding reactionsand/or the nucleotide incorporation reactions described herein. The kitscan include, in one or more containers, the components of nucleotidebinding and/or nucleotide incorporation disclosed herein, including: thesolid surfaces, energy transfer moieties, reporter moieties,nanoparticles, polymerases, nucleotides, target nucleic acid molecules(e.g., a control test target molecules), primers, and/oroligonucleotides.

In the kits, the solid surfaces, energy transfer moieties, reportermoieties, nanoparticles, polymerases, nucleotides, target nucleic acidmolecules, primers, and/or oligonucleotides can be attached to eachother in any combination, and/or be unattached. The kits can includepositive and/or negative control samples.

Additional components can be included in the kit, such as buffers andreagents. For example, the buffers can include Tris, Tricine, HEPES, orMOPS, or chelating agents such as EDTA or EGTA. In another example, thereagents can include monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate. In yet anotherexample, the reagents can include divalent ions, such as Ca²⁺, CaCl₂),Mg²⁺, MgCl₂, Mg-acetate, Mn²⁺, MnCl₂, and the like. The kits can includethe components in pre-measured unit amounts. The kits can includeinstructions for performing the nucleotide binding reactions and/or thenucleotide incorporation reactions. Where the kit is intended fordiagnostic applications, the kits may further include a label indicatingregulatory approval for the diagnostic application.

EXAMPLES

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. In some cases, the compositions and methods of thisinvention have been described in terms of embodiments, however theseembodiments are in no way intended to limit the scope of the claims, andit will be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain components which are both chemically andphysiologically related may be substituted for the components describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 1

Synthesis of Nucleotide Tetraphosphate Molecules Labeled with Alexa Dyes

The synthesis scheme of amino-dN tetraphosphate is illustrated in scheme1 using amino-dG4P as an illustrative example. Amino-attached dA4P, dC4Pand dT4P were synthesized by the same method.

1.) Synthesis of Compound 2

Compound 1 (678 mg, 2 mmol) was suspended in trimethyl phosphate (5 mL)and cooled to 0° C. POCl₃ (280 μL) was added to the stirred mixtureunder argon. The mixture was warmed up and stirred at room temperatureovernight. The reaction was quenched by adding slowly 4 mL of TEABbuffer (1 M) at 0° C. Triethylamine was added to adjust to pH 7. Thesolvent was evaporated and the residue was purified by columnchromatography on silica gel, eluting with 10% H₂O/CH₃CN. Afterevaporation of the solvent, the solid was dissolved in water. The pH ofthe solution was adjusted to pH 7 with TEAB buffer (1 M), followed bycoevaporation with methanol. Yield: 400 mg of compound 2.

2.) Synthesis of Compound 3

The sodium salt of dGTP (20 mg) was converted into its triethylammoniumsalt by passing a triethylammonium resin and dried in high vacuum.Compound 2 (42 mg) was dissolved in 2 mL of dry DMF. carbonyldiimidazole(CDI) (65 mg) was added and the solution was stirred for 4 hours at roomtemperature, followed by the addition of anhydrous methanol (18 μL) andstirred for a further hour. The dried dGTP triethylammonium salt wasdissolved in dry DMF (2 mL), and to this solution was added the preparedphospoimidazolate solution of 2 under argon. The mixture was stirredunder argon overnight. Triethylamine (1 mL) was added and stirred for 4hours. The solvent was evaporated, washed with CHCl₃, dissolved in waterand purified by sephadex A-25 DEAE ion exchange chromatography, elutingwith a linear gradient of 0.05 M to 0.6 M TEAB buffer. Aftercoevaporation with methanol and lyophilization, ca. 5 mg of compound 3was obtained. The reaction was checked by TLC(Dioxane/IPA/H₂O/NH₄OH=40/20/40/36).

3.) Synthesis of Amino-Attached dA4P (4), dC4P (5) and dT4P (6)

These compounds were synthesized by the same method as described foramino-dG4P (3).

4.) Labeling Amino-dGP4 with Alexa Dyes

A solution of amino-dG4P (3) (0.5 mg) in DMF-water (2:1, 300 μL) wasmixed with 50 μL of saturated sodium bicarbonate solution. To thissolution was added the Alexa dye SE (2 mg). The solution was stirred atroom temperature until the completion of the reaction (ca. 1 hour). Theproduct was purified by column chromatography on sephadex LH-20, elutingwith water. The desired fraction was concentrated to ca. 300 μL andstored at −20° C.

The Alexa dye SE used includes AF633 SE, AF647 SE, AF660 SE, AF680 SE,AF700 SE and AF750 SE.

5.) Labeling Amino-dAP4, Amino-dC4P and Amino-dT4P with Alexa Fluor Dyes

These amino-dN tetraphosphates were labeled with Alexa dyes by the samemethod as described in procedure 4.

Example 2

Preparing PEG and Biotin-Streptavidin Coated Surfaces

Low-Density Streptavidin Coating

Low density streptavidin layers were coated on the surface of glasscoverslips using a flowcell. PEG/PEG-biotin coated glass cover slips(MicroSurfaces, Inc., Minneapolis, Minn.) were assembled into 8-lanereaction chambers with laser-cut 3M double-sided adhesive and customfabricated plastic superstructures with inlet/outlet ports for fluidaddition. The surface was wetted by flowing 1 milliliter of TBSBsolution which contains Tris-buffered saline (50 mM Tris, pH 7.5, 150 mMNaCl) and 0.5% bovine serum albumin (Sigma, catalog #A8577). 250microliters of 1% BSA/TBS (50 mM Tris, pH 7.5, 1% BSA) was flowed acrossthe chip and allowed to incubate at room temperature for 5 minutes. Thesurface was coated with streptavidin by flowing 100 microliters of 60 pMstreptavidin, (Zymed, Cat #43-4302) diluted in TBSB, and incubating for30 minutes at room temperature. The lanes were washed with 1 milliliterof TBSB and passivated for a second time with 250 microliters of 1%BSA/TBS-biotinylated DNA, in the form of a self-annealing 5′-overhanginghairpin molecule, was diluted to 10-100 pM in 1% BSA/TBS and 100microliters was flowed into the reaction chamber and incubated 30minutes at room temperature. The lanes were washed with 1 milliliter ofTBSB. The density of the DNA bound to the low densityPEG-biotin-streptavidin coated glass surface was imaged using totalinternal reflection microscopy (TIRF) and a 633 nm laser.

High-Density Streptavidin Coating

High density streptavidin layers were coated on the surface of glasscoverslips using a flowcell. PEG/PEG-biotin coated cover slips(MicroSurfaces, Inc., Minneapolis, Minn.) were assembled into 8-lanereaction chambers with laser-cut 3M double-sided adhesive and customfabricated plastic superstructures with inlet/outlet ports for fluidaddition. The surface was wetted by flowing 1 milliliter of TBSBsolution which contains Tris-buffered saline (50 mM Tris, pH 7.5, 150 mMNaCl) containing 0.5% bovine serum albumin (Sigma, catalog #A8577). 250microliters of 1% BSA/TBS (50 mM Tris, pH 7.5, 1% BSA) was flowed acrossthe chip and allowed to incubate at room temperature for 5 minutes. Thesurface was coated with streptavidin by flowing 100 microliters of 200μg/ml streptavidin, (Zymed, Cat #43-4302) diluted in TBSB, andincubating for 10 minutes at room temperature. The lanes were washedwith 1 milliliter of TBSB and passivated for a second time with 250microliters of 1% BSA/TBS. Biotinylated DNA, in the form of aself-annealing 5′-overhanging hairpin molecule, was diluted to 10-100 pMin 1% BSA/TBS and 100 microliters was flowed into the reaction chamberand incubated 30 minutes at room temperature. The lanes were washed with1 milliliter of TBSB. The density of the DNA bound to the high densityPEG-biotin-streptavidin coated glass surface was imaged using totalinternal reflection microscopy (TIRF) and a 633 nm laser.

Example 3

Linking Chemistries for Attaching Nanoparticles with Polymerases

Preparing Phosphorothiolated Phi29 Polymerases

Phi29 polymerase protein, comprising the protein kinase A recognitionsequence LRRASLG (SEQ ID NO:19) at the N-terminus (SEQ ID NO:7), wasincubated with kinase and ATP-γS to form a phosphorothioate functionalgroup on the serine residue of the recognition sequence.

Modifying the Nanoparticles with Adipic Dihydrazide

C8 Nanoparticles having outer shells which are pre-modified withmethoxy-terminated PEG were obtained from Molecular Probes. Thesenanoparticles have residual carboxylate functional groups. 300 μl of 4.1μM the nanoparticles were buffer exchanged into 100 mM MES, 300 mM NaCl,pH 5.5 using ultrafiltration (VivaSpin 100K MWCO spin filters). Thereaction was started by adding: 260 μl of 4.08 μM buffer exchangednanoparticles, 10.6 μl of 20 mM adipic dihydrazide (dissolved in water)and 13.5 μl of 10 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride dissolved in water). 25 minutes after the start of thereaction another 13.5 μl aliquot of 10 mM EDC was added to the reactionmix. After two hours incubation at room temperature, the reaction mixwas concentrated by ultrafiltration (VivaSpin 100K MWCO) then washedthree times with 200 μl of 100 mM MES, 300 mM NaCl, pH 7.5 using thesame ultrafiltration unit. The nanoparticles have hydrazide functionalgroups.

Reacting the Nanoparticles with Iodoacetic Acid

The nanoparticles (having hydrazide reactive groups) were modified withiodoacetic acid. The following reagents were added: 185 μl of 3.98 μMhydrazide-modified nanoparticles, 14.70 of 10 mM iodoacetic acid (sodiumsalt, dissolved in water) and 10 μl of 10 mM EDC (dissolved in water).25 minutes after the start of the reaction another 10 μl aliquot of 10mM EDC was added to the reaction mix. The reaction mix was allowed toincubate at room temperature, in the dark for three hours. Afterincubation, the reaction mix was concentrated by ultrafiltration andwashed 5× 200 μl with 100 mM MES, 300 mM NaCl, pH 5.5 also usingultrafiltration. The nanoparticles have iodoacetyl functional groups.

Attaching Iodoacetyl Nanoparticles with Phi29 Polymerases

The phosphorothioated phi29 polymerase was buffer exchanged into 100 mMMES, 300 mM NaCl, pH 5.5 using a NAPS column (GE Healthcare). For theconjugation reaction, 392 μl of 13.41M phosphorothioated phi29polymerase was added to 95 μl of 2.73 μM iodoacetyl nanoparticles. Thereaction mix was allowed to incubate overnight at room temperature inthe dark. The reaction mix was concentrated to approximately 30 μl thenpurified over a SUPERDEX 200 (GE Healthcare) 8 mm×5.5 cm column (2 mLdisposable column from Thermo Scientific) using 100 mM TRIS, 300 mMNaCl, pH 7.5 as the elution buffer. Three fractions were collected andassayed for concentration, extension activity and template binding.

Materials:

Hairpin oligonucleotide 221 sequence:

(SEQ ID NO: 24) 5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACCXGC-3′where X=fluorescein dT.Hairpin oligonucleotide ALEXA FLUOR-647-labeled 199 sequence:

(SEQ ID NO: 25) 5′-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACCX-3′where X=ALEXA FLUOR 647-dC1× extension buffer: 50 mM Tris (pH 8), 50 mM NaCl, and 10 mM MgCl₂.Activity Assay

A 150 nM master mix solution of a labeled hairpin oligonucleotide 221was prepared by diluting the appropriate quantity of a 50 μM stocksolution with extension buffer (50 mM TRIS, pH 8, 50 mM NaCl, 10 mMMgCl₂). 450 μl of a master mix was prepared for each sample beingtested.

The conjugate being tested was diluted in 450 μl of the master mix suchthat the final concentration of the conjugate is in the range of 10 nMto 50 nM. The positive control samples of free PKAΦ29 were similarlydiluted. The sample solution was deposited in four microtiter platewells, at 100 μl/well.

The microtiter plate was placed in a plate reader (Molecular Devices,SpectraMax M5) and set up to monitor the fluorescence as function oftime (excitation 490 nm, emission 535 nm, cutoff filter 515 nm). Justprior to starting the plate reader, 2 μl of 1 mM dATP was added to eachof two microtiter wells to start the extension reaction. The other twomicrotiter wells with sample represent no extension controls. The platewas read for an hour or until the samples reached saturation. Theresults indicate that phi29 polymerase, attached to nanoparticles, canincorporate nucleotides.

Binding Assay

Each sample to be tested was diluted to 20 nM in 650 μl of extensionbuffer. 50 μl of the sample was pipetted into each well of the top rowof a microtiter plate.

A 2 μM solution of an ALEXA FLUOR-labeled hairpin oligonucleotide JX338was prepared by dissolving the appropriate amount of stockoligonucleotide in extension buffer. 140 μl of each sample to be testedwas prepared. The hairpin primer/template solution was pipetted into thefirst well of the second row in the microtiter plate. Into the remaining11 wells of the second row of the microtiter plate, 70 μl of extensionbuffer was pipetted. 70 μl of the hairpin primer/template was removedfrom the first well of the second row and mixed with the extensionbuffer in the second well. 70 μl from the second well was removed andmixed with the extension buffer in the third well. The serial dilutionwas prepared up to the last well in row two.

50 μl of the primer/template was transferred from each well of row twointo 50 μl of the sample in each well of row one.

The microtiter plate was placed on the plate reader which was set tomeasure fluorescence at 605 nm and 670 nm with excitation at 450 nm. Theresults showed an increase in FRET acceptor signal with an increase inthe amount of the labeled oligonucleotide-199, or a decrease in FRETdonor signal.

Example 4

Preparing Nanoparticles Attached with His-Tagged Polymerases

Materials:

Hairpin ALEXA FLUOR 647 labeled-oligonucleotide 192 sequence:

(SEQ ID NO: 26) 5′-TTTTTTTGCCCCCAGGGTGACAGGTTTTTCCTGTCACCC-3′where the 192 oligo is labeled at the 3′ end with ALEXA FLUOR 647.Hairpin ALEXA FLUOR 647 labeled-oligonucleotide 199 sequence:

(SEQ ID NO: 25) 5′-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACCX-3′where X=ALEXA FLUOR 647-dC.Hairpin fluorescein labeled-oligonucleotide 221 sequence:

(SEQ ID NO: 24) 5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACCXGC-3′where X=fluorescein dT.Hairpin ALEXA FLUOR 647 labeled-oligonucleotide 229 sequence:

(SEQ ID NO: 27) 5′-TTTTTGCGGGTGACAGGTTTTTCCTGTCACCC-3′where the 229 oligo is labeled at the 3′ end with ALEXA FLUOR 647.

1× extension buffer: 50 mM Tris (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, and0.5 mM MnCl₂.

Preparing Nanoparticles Attached with Phi29 Polymerase

300 μL, of a stock solution of His-tagged phi29 polymerase (SEQ ID NO:8)(56 μM) which is exonuclease minus (flexible linker: GGGGSGGGGSAAAGSAA,SEQ ID NO:20) (stock solution in: 10 mM Tris (pH 7.5) buffer with 100 mMNaCl, 1 mM DTT, 0.5% Tween-20, 0.1 mM EDTA and 50% v/v glycerol) wasbuffer exchanged into 100 mM Tris (pH 7.5) buffer with 300 mM NaCl usingan NAP-5 column.

C8 Nanoparticles (160 μL, 4.9 μM in 50 mM borate buffer pH 8.0) wasconcentrated to approximately 30 μL by ultrafiltration (VivaSpin, at100K MWCO0, and mixed with the buffer exchanged phi29 polymerase (440μL, 26.9 μM in 100 mM Tris (pH 7.5) buffer with 300 mM NaCl n a 1:15molar ratio (nanoparticle to polymerase). The resulting solution wasincubated overnight at 4° C., concentrated to ˜30 μL by ultra-filtrationwith a 100K MWCO VivaSpin centrifugal concentrator, further purified onSUPERDEX 200 column using 100 mM Tris (pH 7.5) buffer with 300 mM NaClas the eluent.

The conjugated nanoparticle-phi29 was assayed to determine nucleotideincorporation activity and DNA binding by detecting FRET signals. Theincorporation reaction contained: 1× extension buffer, 10 nMnanoparticle-phi29 conjugates (or non-conjugated phi29 as a control),150 nM oligonucleotide 221, and 20 dATP.

The results indicate that phi29 polymerase, attached to nanoparticles,can incorporate nucleotides.

The binding reactions contained: 1× extension buffer, C8nanoparticles-phi29 conjugates (or phi29 non-conjugated),oligonucleotide 199, and dATP. The binding reactions were seriallydiluted. The results showed an increase in FRET acceptor signal with anincrease in the amount of the labeled oligonucleotide-199, or a decreasein FRET donor signal.

Preparing GST-Nanoparticles Attached with Phi 29 Polymerase

C8 Nanoparticles (50 μL, 3.5 μM in 50 mM borate buffer pH 8.0) wasdiluted with 100 μL of 100 mM Tris buffer pH 7.5 with 300 mM NaCl andconcentrated to ˜20 μL by ultrafiltration (VivaSpin, 100K MWCO). Theconcentrated nanoparticle solution was mixed with His-tagged-GST (184μL, 19 μM in 50 mM Tris pH7.5 with 200 mM NaCl) in a 1:20 molar ratio(nanoparticle to His-tagged-GST). The resulting solution was incubatedat room temperature for 5 hours. Phi29 polymerase (SEQ ID NO:8) (60 μL,14.5 μM in 100 mM Tris (pH 7.5) buffer with 300 mM NaCl) was added tothe nanoparticles in a 5:1 molar ratio (phi29 to nanoparticle). Theresulting solution was incubated overnight at 4° C., concentrated to ˜30μL by ultra-filtration with 100K MWCO VivaSpin centrifugal concentrator,purified on a SUPERDEX 200 column using 100 mM Tris (pH 7.5) buffer with300 mM NaCl as the eluent.

The conjugated GST-nanoparticle-phi29 were assayed to determine templateextension activity and DNA binding by detecting FRET signals. Theincorporation reaction contained: 1× extension buffer, 10 nMnanoparticle-phi29 conjugates (or non-conjugated phi29 as a control),150 nM oligonucleotide 221, and 20 dATP.

The results indicated that phi29 polymerase, attached to GST-treatednanoparticles, can incorporate nucleotides.

The binding reactions contained: 1× extension buffer, C8nanoparticles-phi29 conjugates (or phi29 non-conjugated),oligonucleotide 199, and dATP. The binding reactions were seriallydiluted. The results showed an increase in FRET acceptor signal with anincrease in the amount of the labeled oligonucleotide-199, or a decreasein FRET donor signal.

Preparing UDG-Ugi-Nanoparticles Attached with Phi29 Polymerase

His tagged UDG protein (uracil DNA glycosylase) (500 μL, 27 mM in 30 mMTris buffer (pH 7.5) with 200 mM NaCl) was mixed ugi (uracil-DNAglycosylase inhibitor) (50 μL, 347 μM in 30 mM Tris buffer (pH 7.5) with200 mM NaCl) in 1:1.2 molar ratio (His-tagged-UDG to ugi protein), andincubated at 4° C. overnight.

C8 Nanoparticles (140 μL, 4.9 μM in 50 mM borate buffer pH 8.0) wasdiluted by 200 μL of 100 mM Tris buffer (pH 7.5) with 300 mM NaCl andconcentrated to ˜30 μL by ultrafiltration (VivaSpin, 100K MWCO). Theconcentrated nanoparticle solution was mixed with the His-tagged-UDG-ugiprotein conjugate (550 μL, 24.7 μM in 30 mM Tris buffer (pH 7.5) with200 mM NaCl) in a 1:20 molar ratio (nanoparticle to His-tagged-UDG-ugi)to prepare the UDG-ugi-nanoparticles. The resulting solution wasincubated at room temperature for 5 hours.

The phi29 polymerase (SEQ ID NO:8) was added (220 μL, 15.4 μM in 100 mMTris (pH 7.5) buffer with 300 mM NaCl) in a 1:5 molar ratio(UDG-ugi-nanoparticle to phi29). The resulting solution was incubatedovernight at 4° C., concentrated to ˜30 μL by ultra-filtration with 100KMWCO VivaSpin centrifugal concentrator, and purified on a SUPERDEX 200column using 100 mM Tris (pH 7.5) buffer with 300 mM NaCl as the eluent.

The conjugated UDG-ugi-nanoparticle-phi29 was assayed to determinetemplate extension activity and DNA binding by detecting FRET signals.The incorporation reaction contained: 1× extension buffer, 10 nMnanoparticle-phi29 conjugates (or non-conjugated phi29 as a control),150 nM oligonucleotide 199, and 20 μM dATP.

The results showed that phi29 polymerase, attached to UDG/ugi-treatednanoparticles, can incorporate nucleotides.

The binding reactions contained: 1× extension buffer, C8nanoparticles-phi29 conjugates (or phi29 non-conjugated),oligonucleotide 199, and dATP. The binding reactions were seriallydiluted. The results showed an increase in FRET acceptor signal with anincrease in the amount of the labeled oligonucleotide-199, or a decreasein FRET donor signal.

Preparing BSA-Nanoparticles Attached with Phi29 Polymerase

Bovine serum albumin (BSA) (20 mg, catalog # B4287, Sigma) was dissolvedin 2 mL deionized water. The BSA solution (200 μL, 10 mg/mL in H₂O wasmixed with DTT (8 μL, 1M), and incubated at room temperate overnight.The resulting solution was purified on an NAP-5 column using deionizedwater as the eluent.

A1 Nanoparticles (100 μL, 1.0 μM in 50 mM Tris buffer (pH 8)) wasdiluted by 100 μL of 100 mM Tris buffer (pH 7.5) with 300 mM NaCl andconcentrated to ˜30 μL by ultrafiltration (VivaSpin, 100K MWCO).

The concentrated nanoparticle solution was mixed with DTT (1.0 μL, 100mM), and with the above-described BSA solution (27 μL, 75.8 μM indeionized water) in a 1:20 molar ratio (nanoparticle to BSA). Theresulting solution was incubated at room temperature overnight,concentrated to ˜30 μL by ultra-filtration 100K MWCO VivaSpincentrifugal concentrator.

The concentrated nanoparticle-BSA solution was mixed with the phi29polymerase (SEQ ID NO:8) (48 μL, 20.8 μM in 100 mM Tris (pH 7.5) bufferwith 300 mM NaCl) in a 1:10 molar ration (BSA-nanoparticles to phi29).The resulting solution was incubated overnight at 4° C., concentrated to˜30 μL by ultra-filtration with 100K MWCO VivaSpin centrifugalconcentrator, and purified on a SUPERDEX 200 column using 100 mM Tris(pH 7.5) buffer with 300 mM NaCl as the eluent.

The conjugated BSA-nanoparticle-phi29 was assayed to determine templateextension activity and DNA binding by detecting FRET signals. Theincorporation reaction contained: 1× extension buffer, 10 nMnanoparticle-phi29 conjugates (or non-conjugated phi29 as a control),150 nM oligonucleotide 229, and 20 μM dATP.

The results showed that phi29 polymerase, attached to BSA-treatednanoparticles, can incorporate nucleotides.

The binding reactions contained: 1× extension buffer, C8nanoparticles-phi29 conjugates (or phi29 non-conjugated),oligonucleotide 229, and dATP. The binding reactions were seriallydiluted. The results showed an increase in FRET acceptor signal with anincrease in the amount of the labeled oligonucleotide-229, or a decreasein FRET donor signal.

Example 5

Nucleotide Polymerization Using Polymerases Attached to Nanoparticles

Materials

Nanoparticle shapes: A1 are spherical, and A2 and A4 are rod-shaped. Thespherical nanoparticles are about 8 nm in diameter, and the rod-shapedones are about 5×12 nm (width×length). These nanoparticles have ligandcoatings which include: L-carnosine; dipeptides (e.g., His-Leu andGly-His); 4-aminobenzophenone; citric acid; glycine;tris(hydroxymethyl)phosphine; and amino-dPEG24-acid.

The nanoparticles were reacted with HRP, BSA, biotin, and conjugatedwith one of three different phi29 polymerases: HP1, HP1-Q380A orHP1-S388G.

HP1 is a 6× His-tagged phi29 polypeptide (‘6× His’ disclosed as SEQ IDNO: 63) which is exonuclease-minus (SEQ ID NO:9). HP1-Q380A is a 6×His-tagged phi29 mutant polypeptide (‘6×His’ disclosed as SEQ ID NO: 63)which is exonuclease-minus (SEQ ID NO:10). HP1-S388G is a 6× His taggedphi29 mutant polypeptide (‘6×His’ disclosed as SEQ ID NO: 63) which isexonuclease-minus (SEQ ID NO:11).

Attaching Nanoparticles Attached with Polymerases

Horseradish peroxidase (HRP; Invitrogen; Cat #01-2001) reductionreaction: 3 mg of HRP was reacted with 150 mg of Cleland's REDUCTACRYLReagent (VWR; Cat #80056-208) in 600 μl of 50 mM sodium borate buffer,pH 8.2 for 45 minutes at room temperature. The reaction was filteredthrough a Micro Bio-Spin Empty Column (Bio-Rad; Cat #732-6204). 360 pmolof spherical (A1) or rod-shaped (A2 or A4) nanoparticles (1 eq.) wereadded in 50 μl of 50 mM sodium borate buffer, pH 8.2 containing 5 μL of10% BSA (Invitrogen; Cat #P2489) for 1 hour at room temperature. Thereaction mixture was concentrated using a VivaSpin 500 100 KDa MWCOultrafiltration unit (VWR; Cat #14005-008) and washed (5 times) with 50mM sodium borate buffer (pH 8.2). 3 mg of LC-sulfo-NHS-Biotin (MolecularBiosciences; Cat #00598) was added in 300 μl of 50 mM sodium boratebuffer, pH 8.2 for 30 min at room temperature. The reaction was filteredand washed again as above (5 times), diluted with 100 μl of sodiumborate buffer containing 300 mM NaCl (final concentration in a finalreaction volume). Phi29 polymerase (HP1 or HP1-Q380A (15 eq.) was addedand incubated at 4° C. overnight. Reaction mixtures were purified usinga SUPERDEX column (VWR; Cat #95017-068) eluting with a borate buffercontaining 300 mM NaCl and concentrated to 1-2 μM of conjugationproducts using VivaSpin 500 100 KDa MWCO filters and centrifugation at6,000×G.

Assay: Confirming Nanoparticles are Conjugated with Polymerases

Assays were performed to confirm that the phi29 polymerases wereattached to the nanoparticles. The assay included 250 nM of ALEXA FLUOR647 labeled oligonucleotide:

(5′-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACC-3′-ALEXA FLUOR 647) (SEQ ID NO:64) and 40 nM of the nanoparticle-polymerase conjugates in 50 mM Tris,50 mM NaCl and 10 mM MgCl₂. The reaction was excited at 450 nm andemission (e.g., FRET) was detected as a ratio of intensities at 605/670(nanoparticle emission/ALEXA FLUOR 647 emission). Control nanoparticleswere reacted with HRP, BSA, biotin, and ALEXA FLUOR 647, but no phi29polymerase.

The results showed that the control nanoparticles exhibit a higherintensity peak compared to the nanoparticles conjugated with phi29polymerase and dye-labeled oligonucleotides at the same concentration,and the signal intensity peaks at 670 nm. This demonstrates that thenanoparticles are bound with the phi29 polymerase and with the ALEXAFLUOR 647-labeled oligonucleotide.

Assay: Nucleotide Incorporation

Assays were performed to determine if the polymerases, which areattached to the nanoparticles, could incorporate nucleotides. The assayincluded 150 nM of a hairpin oligonucleotide, fluorescein-labeledoligo-221:

(5′-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACC(fluorescein-T)GC-3′) (SEQ IDNO:28), and 40 nM of the nanoparticle-polymerase conjugates, 20 μM dATPin 50 mM Tris, 50 mM NaCl, and 10 mM MgCl₂ buffer. The reaction wasexcited at 490 nm and emission was detected at 525 nm. Controlnanoparticles were reacted with HRP, BSA, biotin, and ALEXA FLUOR 647,and no phi29 polymerase. The results showed that the controlnanoparticles exhibit baseline intensity fluorescence levels compared tonanoparticles bound with phi29 polymerase and dye-labeledoligonucleotides. These results demonstrate that phi29 enzyme conjugatedwith a nanoparticle retains its nucleotide incorporation activity.

Assay: Nucleotide Incorporation and DNA Extension

Assays were performed to determine if the polymerases, which areattached to the nanoparticles, could polymerize nucleotides. The assayincluded 50 mM Tris (pH 7.0), 2 mM MnCl₂, 62.5-70 mM NaCl (from thevarious nanoparticle-polymerase conjugate stocks), 0.5% BSA, 1 μM eachdNTP, 50 nM duplex (primer Top: 5′-GGTACTAAGCGGCCGCATG-3′ (SEQ ID NO:29)with template C6gOV: 5′-TAAAGCCCCCCCATGCGGCCGCTTAGTACC-3′ (SEQ ID NO:30)or template T6gOV: 5′-TAAAGTTTTTTCATGCGGCCGCTTAGTACC-3′) (SEQ ID NO:31),and 100 nM of HP1 phi29 polymerase (no nanoparticles), 100 nM A1/HRP-HP1(A1 spherical nanoparticles conjugated with phi29 polymerase), or 100 nMA4/HRP-HP1 (A4 rod-shaped nanoparticles conjugated with phi29polymerase). The reaction was initiated with the addition of dNTPs (1μM) including dA4P labeled at the terminal phosphate group with oneALEXA FLUOR 647, dG4P labeled at the terminal phosphate group with ALEXAFLUOR 680, and dCTP labeled at the nucleo-base with Cy5 dye (GEHealthcare Biosciences; catalog #PA55021). The reaction was quenchedwith EDTA and analyzed by electrophoresis in a 20% 7M urea denaturinggel followed by fluorescence imaging. The results showed extensionproducts from phi29 polymerase in all three forms (unbound; bound tospherical nanoparticles (A1); and bound to rod nanoparticles (A4)). Theresults also showed extension products produced by phi29 polymerase,bound to nanoparticles, and incorporating fluorescent dye labeleddeoxynucleotide tetraphosphate molecules (dA4P and dG4P).

Assay: Nucleotide Incorporation and DNA Extension

Assays were performed to determine if the polymerases, which areattached to the nanoparticles, could polymerize nucleotides. The assayincluded 50 mM Tris (pH 7.0), 2 mM MnCl₂, 42.5-167.5 mM NaCl (fromvarious nanoparticle-polymerase conjugate stocks), 0.5% BSA, 1 μM eachdNTP, 100 nM duplex

(primer Top: 5′-GGTACTAAGCGGCCGCATG-3′ (SEQ ID NO:29) with templateC6gOV: 5′-TAAAGCCCCCCCATGCGGCCGCTTAGTACC-3′ (SEQ ID NO:30) or templateA6A: 5′-GGTACTAAGCGGCCGCATGAAAAAAA-3′) (SEQ ID NO:32), and 200 nM of HP1phi29 polymerase (no nanoparticles) or 200 nM of A2/HRP-HP1 (rod-shapednanoparticles conjugated with phi29 polymerase). The reaction wasinitiated with the addition of 1 μM of dNTPs, including dCTP labeled atthe nucleo-base with Cy5 dye (GE Healthcare Biosciences; catalog#PA55021) in combination with dG4P labeled at the terminal phosphategroup with ALEXA FLUOR 680 or with dGTP. For the A6A template, thereaction was conducted in the presence of dU4P labeled at the terminalphosphate group with ALEXA FLUOR 680 and labeled at the nucleo-base withALEXA FLUOR 647. The reactions were quenched with EDTA and analyzed bygel electrophoresis in a 20% 7M urea denaturing gel followed byfluorescence imaging. The results showed extension products from phi29polymerase in four forms: (1) unbound HP1 polymerase, (2) HP1 polymerasebound to A2 rod-shaped nanoparticles (A2-HP1), (3) HP1 polymerase mutantQ380A bound to A2 rod-shaped nanoparticles A2-HP1-Q380A), and (4) HP1polymerase mutant S388G bound to A2 rod-shaped nanoparticles(A2-S388G-Phi29). The results also showed extension products produced byphi29 polymerase bound to nanoparticles and incorporatingdeoxynucleotide tetraphosphate molecules (dG4P) and fluorescent-dyelabeled deoxynucleotide tetraphosphate molecules (dG4P-Alexa 680).Detecting FRET Signals in a Single Molecule Assay

Chambered glass cover slips were prepared to facilitate injection andmultiple experiments data collection from several chambers using asingle slide. The PEG-neutravidin glass coverslips were functionalizedas described by Taekjip Ha (2002 Nature 419:638-641) but usingneutravidin instead of streptavidin. Duplexes of primer/template strandswere prepared by reacting 1 μM of the template and 1 μM of the primerstrands in 1× Duplexing buffer (50 mM Tris (pH 7.2), 10 mM NaCl).

Reaction 1: Primer: (SEQ ID NO: 33) 5′-TGATAGAACCTCCGTGT-3′ Template:(SEQ ID NO: 34) 5′-GGAACACGGAGGTTCTATCATCGTCATCGTCATCGTCATCG-3′;Reactions 2 and 3: Primer: (SEQ ID NO: 35) 5′-GGTACTAAGCGGCCGCATG-3′Template: (SEQ ID NO: 36) 5′-TTTTACCCATGCGGCCGCTTAGTACC-3′; Reaction 4:Primer: (SEQ ID NO: 37) 5′-GGTACTAAGCGGCCGC-dd-3′ Template: (SEQ ID NO:38) 5′-TTTTACCCATGCGGCCGCTTAGTACC-3′.

10 nM of the nanoparticles (which were conjugated with phi29 polymerasemutant Q380A) were reacted with 300 nM of the DNA primer/template duplexon ice for 30 minutes in 1× pre-complexing buffer (50 mM Tris (pH 7.2),100 mM NaCl) in a total volume of 100 μL. This reaction forms the binarycomplex of nanoparticle/polymerase bound with template/primer.

The binary complex was diluted to a nanoparticle/polymerase (100 pM) andtemplate/primer duplex (3000 pM) to a ratio of 1:30. 100 μL of thediluted binary complex was injected into a chamber and was allowed toimmobilize on the PEG-neutravidin surface for 5 minutes. An extensionmix was injected and the reaction was allowed to occur for 2 minutes,followed by a 200 μL of EDTA and an oxygen scavenging system containingbuffer wash. The extension mix consisted of 50 mM Tris (pH 7.2), 2 mMMnCl₂, 100 mM NaCl, 0.5% BSA and natural dNTPs (dGTP) or dye-labeleddNTPs (dG4P labeled at the terminal phosphate group with ALEXA FLUOR 680and Cy5 base-labeled dUTP) at 1 μM each. The oxygen scavenging systemconsisted of 50 nM protocatechuate-3,4-dioxygenase, 2.5 mMprotocatechuic acid and 1 mM TROLOX(6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid;Hoffmann-LaRoche).

Four separate reactions were performed: Reaction #1 included Cy5base-labeled dUTP (GE Healthcare Biosciences; catalog # PA55022).Reaction #2 included dGTPs and Cy5 base-labeled dUTP (GE HealthcareBiosciences; catalog # PA55022). Reaction #3 included dG4P labeled atthe terminal phosphate group with ALEXA FLUOR 680 and Cy5 base-labeleddUTP (GE Healthcare Biosciences; catalog # PA55022). Reaction #4included dG4P labeled at the terminal phosphate group with ALEXA FLUOR680 and Cy5 base-labeled dUTP (GE Healthcare Biosciences; catalog #PA55022), and the primer having a ddG at the 3′ end (negative control).

The data were collected on the single molecule detection system, whichincluded an ANDOR back-illuminated EMCCD camera (iXonEM), and aninverted Olympus microscope (IX71), with a 100×TIRF objective. Thesamples were excited using a 405 nm laser (Coherent; Cat #1069413) at460 μW, and the data was collected at 100 ms integration time for 2000frames and 3 to 5 consecutive streams were collected by moving to newfields of views (FOVs). The signals were separated using dichroics (535nm, 667 nm) before forming an image on the camera.

FRETAN software (Volkov et al., U.S. Ser. No. 11/671,956) was used toobtain donor and acceptor FRET traces. Custom-designed MATLAB scriptswere used to extract the data and obtain percent FRET or percentactivity data. Only acceptor donor type signals and acceptors with S/Ngreater than 2 were counted for the percent activity numbers.

Example 6

Preparation of Core-Shell Nanoparticle CdSe/4CdS-3.5ZnS Core Synthesis

Cores are prepared using standard methods, such as those described inU.S. Pat. No. 6,815,064, the only change being that the growth is haltedat 535 nm emission. These cores were precipitated and cleaned in thestandard methods and resuspended into hexane for use in the shellreaction.Shell Synthesis:

A 1:1 (w:v) mixture of tri-n-octylphosphine oxide (TOPO) andtri-n-octylphosphine (TOP) was introduced into a flask.Tetradecylphosphonic acid (TDPA) was added to the flask in an amountsuitable to fully passivate the final material, as can be calculatedfrom the reaction scale and the expected final nanoparticle size. Thecontents of the flask were heated to 125° C. under vacuum and then theflask was refilled with N2 and cooled.

Inside the glovebox, a solution of a suitable cadmium precursor (such asdimethylcadmium or cadmium acetate) in TOP was prepared in a quantitysufficient to produce a desired thickness of shell, as can be calculatedby one of ordinary skill in the art. When a zinc shell was also desired,a solution of a suitable zinc precursor (such as diethylzinc or zincstearate) was prepared in TOP in a quantity sufficient to produce thedesired shell thickness. Separately, a solution of trimethylsilylsulfide[(TMS)₂S] in TOP was prepared in a quantity sufficient to produce thedesired shell thickness. Each of these solutions was taken up inseparate syringes and removed from the glove box.

Of the previously prepared core/hexane solution, 17 mL (at an opticaldensity of 21.5 at the band edge) was added to the reaction flask andthe hexane was removed by vacuum; the flask was then refilled with N2.The flask was heated to the desired synthesis temperature, typicallyabout 200 to about 250° C. During this heat-up, 17 mL of decylamine wasadded.

The cadmium and sulfur precursor solutions were then added alternatelyin layer additions, which were based upon the starting size of theunderlying cores. So this means that as each layer of shell material wasadded, a new “core” size was determined by taking the previous “core”size and adding to it the thickness of just-added shell material. Thisleads to a slightly larger volume of the following shell materialneeding to be added for each subsequent layer of shell material.

After a desired thickness of CdS shell material was added, the cadmiumprecursor solution was replaced with the zinc precursor solution. Zincand sulfur solutions were then added alternately in layer additionsuntil a desired thickness of ZnS was added. A final layer of the zincsolution was added at the end, the reaction flask was cooled, and theproduct was isolated by conventional precipitation methods.

Example 7

Exchange Process Using Dipeptide Ligands and Butanol as a Cosolvent

Core/shell nanocrystals (quantum dots) were prepared by standardmethods, and were washed with acetic acid/toluene several times, andsuspended in hexanes. 10 nmol of core/shell nanocrystals were suspendedin 40 mL hexane. This was mixed with 10 mL of a 300 mM solution ofcarnosine and 10 mL of 1 M sodium carbonate solution. n-Butanol (14 mL)was added, and the vessel was flushed with argon. The mixture was mixedvigorously overnight at room temperature. The mixture was then heatedand allowed to cool to room temperature. The aqueous phase was thenremoved and filtered through a 0.2/0.8 micron syringe filter.

Excess carnosine was removed by dialyzing against 3.5 L of 25 mM NaClfor one hour. The solution was concentrated to 1 mL using a 10K MWCO(10,000 molecular weight cut-off) Amicon centricon. A solution was thenprepared with 568 mg of His-Leu dipeptide plus 212 mg of Gly-Hisdipeptide in 9 mL sodium carbonate solution, and this solution wascombined with the aqueous solution of quantum dots. This mixture wasstirred overnight at room temperature. The mixture of water-solublequantum dots was then dialyzed against 3.5 L of 25 mM NaCl for one hour.

To crosslink the peptide ligands (clarify)A solution of 0.5 mM4-aminobenzophenone in ethanol was then added to the aqueous quantumdots mixture, and the mixture was irradiated at 365 nm for 4 hours toeffect reaction of the aminobenzophenone with the surface molecules onthe quantum dots. To this, 5 mmol of THP (tris(hydroxymethyl)phosphine)was added, and the mixture was stirred at RT overnight, to inducecrosslinking. Another 5 mmol of THP was added, and again the mixture wasstirred overnight at RT. Another 5 mmol of THP was added the next day,along with 300 micromoles of PEG1000-COOH. This was mixed overnight atroom temperature, then another 5 mmol of THP was added along with 30mmol of glycine, and the mixture was stirred overnight at RT.

The material was purified by dialysis using the 10K MWCO Amiconcentricon, and was washed with 50 mM borate buffer (pH 9). The finalmaterial was dispersed into 50 mM borate buffer to a final concentrationof 2.5 micromolar for storage.

Example 8

Exchange Process Using Trithiol Ligands

A solution of hydrophobic phosphonate-coated quantum dots in organicsolvent (e.g. toluene, chloroform, etc) with a concentration of betweenabout 0.1 and 10 micromolar quantum dots was prepared. Approximately1000 to 1000000 equivalents of a suitable trithiol ligand was added,optionally as a solution in a suitable organic solvent (e.g. acetone,methanol, etc). The reaction mixture was stirred for 1-48 hours and thenthe solution was basicified by addition of an organic base (e.g.tetramethylammonium hydroxide, tetrabutylammonium hydroxide, etc). Aftera shorter second stirring period, water or aqueous buffer was used toextract the dots with hydrophilic ligands. The aqueous solution waswashed with additional organic solvent (e.g. toluene, chloroform, etc)and purified by filtration.

Example 9

Two-Step Ligand Exchange: Process for Exchanging Phosphonate Ligandswith Sulfonate (Triflate) Ligands

A nanoparticle comprising a core/shell nanocrystal having TDPA ligandson its surface is dissolved in dichloromethane, and excess TMS triflateis added to it. After 1-2 hours at room temperature, analysis indicatesthat the TDPA ligands have been removed, and the nanoparticle remainsdispersed in the solvent. It is dialyzed against dichloromethane using a10K MWCO (10,000 molecular-weight cut-off) dialysis membrane to removeexcess TMS triflate and the TMS-TDPA produced by the reaction of TMStriflate with the TDPA ligands. This produces a solution/suspension ofnanoparticles comprising triflate ligands on the surface ofnanocrystals. These triflate-containing nanoparticles are soluble inmany organic solvents, but may not be readily soluble in hexanes,depending upon the complement of ligands present.

Two-Step Process for Exchanging Sulfonate (Triflate) Ligands with PEGConjugated Dithiol (DHLA) Ligands Using n-Butanol as an IntermediateLigand and DMF as a Co-Solvent

The triflate-containing nanoparticle solution, described above, can becontacted with excess n-butanol in acetonitrile, using DMF as aco-solvent, to provide an intermediate nanoparticle believed to comprisebutanol ligands in place of the triflates which were on thenanoparticle. This intermediate nanoparticle can be isolated from themedium, or it can be further modified without isolation. Thisintermediate nanoparticle is contacted with an excess of a dihydrolipoicacid-PEG conjugate of this formula: where n is 1-100.

The product is a water-soluble, stable nanoparticle. It can be collectedby extraction into a pH 9 buffer, and isolated by conventional methods,including dialysis with a 10K MWCO dialysis filter, or by size exclusion(gel filtration) chromatography.

Two-Step Process for Exchanging Sulfonate (Triflate) Ligands withNucleophilic Reactant Group Containing Ligands Using n-Butanol as anIntermediate Ligand and DMF as a Co-Solvent

The triflate-containing nanoparticle solution from can be contacted withexcess n-butanol in acetonitrile, using DMF as a co-solvent, to providean intermediate nanoparticle believed to comprise butanol ligands inplace of the triflates which were on the nanoparticle. This intermediatenanoparticle can be isolated from the medium, or it can be furthermodified without isolation. To further modify it, it is treated with anew ligand containing at least one nucleophilic reactant group: suitableligands include HS—CH₂—CH₂-PEG; aminomethyl phosphonic acid;dihydrolipoic acid; omega-thio-alkanoic acids, andcarboxymethylphosphonic acid. The mixture is then treated with TMEDA(tetramethylethylene diamine), and monitored until triflate isdisplaced, then the nanocrystal product is extracted into pH 9 bufferand purified by conventional methods.

Process for Exchanging Sulfonate (Triflate) Ligands with CarboxylateFunctionalized Dithiol (DHLA) Ligands

The triflate-containing nanoparticle is contacted with neatdihydrolipoic acid (DHLA) for an hour at room temperature, and is thendispersed into pH 9 buffer and isolated by conventional methods. Thisprovides a nanoparticle having carboxylate groups to provide watersolubility, and having two thiol groups binding the carboxylate to thenanocrystal surface. The product is water soluble and stable in aqueousbuffer. It provides good colloidal stability, and a moderate quantumyield. This composition containing DHLA as a ligand contains freecarboxyl groups which can be used to attach other groups such as a PEGmoiety, optionally linked to a functional group or a biomolecule. Thesame reaction can be performed to replace triflate groups on ananoparticle with thioglycolic acid (HS—CH₂—COOH) ligands. This providesa highly stabilized nanoparticle which produces a high quantum yield,but has lower colloidal stability than the product having DHLA on itssurface.

Process for Exchanging Sulfonate (Triflate) Ligands with Amine Ligands

The triflate-containing nanoparticle is dispersed in dichloromethaneplus hexanes, and an alkylamine is added. Suitable alkylamines arepreferably primary amines, and include, e.g., H₂N—(CH₂)_(r)-PEG(r=2-10), p-aminomethylbenzoic acid, and lysine ethyl ester. After anhour at room temperature, the exchange process is completed, and thenanoparticle product can be isolated by conventional methods.

Process for Pre-Treating Phosphonate Coated Nanocrystals with TolueneAcetic Acid to Remove Impurities Prior to Exchanging with Sulfonate(Triflate) Ligands

TDPA-covered nanocrystals were synthesized which emitted light at 605 nmand had shells of CdS and of ZnS. These when treated with 200,000equivalents of TMS triflate in hexanes did not produce a precipitate.This was attributed to excess TDPA-derived impurities in thenanocrystals. This was alleviated by dissolving the nanocrystals intoluene-acetic acid and precipitating them with methanol, to remove TDPAsalts or related by-products. The resultant TDPA nanocrystals behaved asdescribed above, demonstrating that impurities were causing thenanocrystals to behave differently when made with excess TDPA present,and that those impurities can be removed by precipitation underconditions better suited to dissolving TDPA-related impurities.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals withDithiol (DHLA) Ligands Using Butanol, DMF or Isopropyl Alcohol asDispersants

Three different methods of depositing the DHLA ligands were employed,each of which was considerably more rapid than the classic approachusing non-activated dots. In the first approach, the activated dotpowder was dispersed in butanol and stirred with DHLA, then precipitatedwith hexane and collected in aqueous buffer. In the second approach, theactivated dot powder was dispersed in dimethylformamide (DMF) andstirred with DHLA, then precipitated with toluene and collected inaqueous buffer. In the third approach, the activated dot powder wasstirred as a slurry in neat DHLA, then dispersed in isopropyl alcohol,precipitated with hexane, and collected in aqueous buffer and purifiedwith a filtration membrane.

These three samples, plus a sample derived from non-activated dots werediluted to 60 nM for a colloidal stability challenge, wherein theabsorbance is monitored over the course of days to watch forprecipitation. Samples 1 (butanol-mediated), 2 (DMF-mediated), and 4(classic) all precipitated on day 3 or 4 of the stability challenge, butsample 3 (neat DHLA) lasted twice as long, coming out of solution on day7. HPLC measurements indicated that the DHLA-coated particles producedfrom activated dots showed even less aggregation than the classic DHLAparticles made by the displacement of TOPO or pyridine ligands fromnanocrystals. Thus the invention provided rapid reactions leading toimproved colloidal stability and comparable or lower aggregation levelsthan conventional ligand replacement methods of putting DHLA on ananocrystal. Similar treatment with other thiol ligands likemercaptoundecanoic acid (MUA) or the PEGylated thiol also providedwater-dispersible nanocrystals. Reacting triflate-coated nanoparticleswith MUA or PEG-thiol gave particles which were readily dispersible inwater, indicating that ligand exchange had occurred. The observedquantum yield was over 70% in each case.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals withHydrophilic Phosphonate Ligands

Triflate-coated dots were dispersed in butanol and then stirred withphosphonoacetic acid. Triethylamine was added to form thetriethylammonium salt of both the phosphonate and carboxylatefunctionalities, and then pH 9 aqueous borate buffer was added toextract the hydrophilic particles. The result was a bright orangeaqueous dispersion of quantum dots, with no remaining color observed inthe butanol layer. The particles were purified by centrifugal filtrationand the quantum yield was measured to be 72%. Multiple batches ofparticles were prepared and remained in solution through roomtemperature storage for at least eight weeks. The same method can besuccessfully employed with DHLA, MUA, and PEGylated thiol ligands.

Process for Exchanging Activated (Sulfonate Coated) Nanocrystals with aVariety of Hydrophilic Phosphonate Ligands Via Biphasic Exchange

Using a biphasic exchange method, dispersing the quantum dots in organicsolvents such as chloroform and the exchangeable ligands in aqueoussolution, quantum dots were made water soluble and stable after ligandexchange with N,N-Bis(phosphonomethyl)glycine (1) or phosphonoaceticacid (2). In a typical bi-phasic ligand exchange experiment, 1 nmol ofquantum dots were dispersed in 1 mL of chloroform and placed in a vialwith 2 mL of 300 mM phosphonic acid in basic buffer and the mixture wasrapidly stirred at room temperature for 2 days. Quantum yields as highas 53% were achieved; however the quantum yields achieved were dependenton core-shell batch employed, probably as a result of variable amountsof long-chain alkyl phosphonates remaining on the nanocrystal surfacepost-ligand exchange. This demonstrated that complete removal of TDPAfrom nanocrystals is important for successful modification of thesurface. Though the dots were rendered water stable by the abovephosphonate-containing ligands, they were not successfully modified withPEG2000-diamine using standard EDC condensation chemistry.

Nanocrystals coated with compounds 1, 2, or 3 were readily prepared bythis method, as well as nanocrystals having a mixture of compounds 1 and2, or 1 and 3, or 2 and 3. In each case, the nanocrystals were stable,bright and water-soluble. Using mixed ligands, it was found thatPEGylation (with PEG2000-diamine using standard EDC condensationchemistry) could be achieved with these phosphonate-containing ligandsto produce highly stable, bright, water soluble nanoparticles. Thesenanoparticles can be further stabilized by at least partiallycross-linking the ligands using a diamine such as putrescine,cadaverine, 1,2-diaminoethane, bis(hexamethylene)triamine, PAMAMdendrimer, and cystamine.

Two-Step Ligand Exchange Process with Tridentate Thiol Ligands

Triflate exchange step was performed following the procedure describedabove. Next, the triflate nanoparticles were dispersed in organicsolvent (e.g. toluene, chloroform, etc) with a concentration of betweenabout 0.1 and 10 micromolar quantum dots. Approximately 1000 to 1000000equivalents of a suitable tridentate thiol ligand was added, optionallyas a solution in a suitable organic solvent (e.g. acetone, methanol,etc). The reaction mixture was stirred for 1-48 hours and then thesolution was basicified by addition of an organic base (e.g.tetramethylammonium hydroxide, tetrabutylammonium hydroxide, etc). Aftera shorter second stirring period, water or aqueous buffer was used toextract the dots with hydrophilic ligands. The aqueous solution waswashed with additional organic solvent (e.g. toluene, chloroform, etc)and purified by filtration.

Example 10

Functionalized Ligands on Nanoparticles

General Core Reaction Procedure

Into a 25 mL 3 neck flask with 14/20 joints, 1.575 g of >99%tri-n-octylphosphine oxide (TOPO) was weighed. To this, 1-1000micromoles of a bi-functional phosphonate ligand was added. A stir barwas added to this flask. The flask was connected to an inert atmospheremanifold and evacuated thoroughly, then refilled with nitrogen. Asolution of a suitable cadmium salt in tri-n-octylphosphine (TOP) wasprepared with a concentration of 0.5 mol Cd per kg solution. A desiredamount of cadmium as required for growth of nanoparticles of a desiredsize was extracted from this solution, diluted with 0.9 mL of additionalTOP, and added to the flask. The flask was stirred and heated to˜200-350° C. under nitrogen flow. A 1 molar solution of selenium in TOPwas prepared and a desired amount as required for growth ofnanoparticles of a desired size was added to the solution, optionallywith addition of a reaction promoter to achieve desired levels ofparticle nucleation. One minute after the reaction was initiated byadding these final reagents, a 20 microliter sample was removed from thereaction, mixed with 5 mL of hexane, and an emission spectrum wascollected. This aliquot removal and measurement process was repeatedafter 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 minutes. After 14 minutes, thereaction was rapidly cooled and the products were isolated by methodsunderstood in the art.

Control Core Reaction with Tetradecylphosphonic Acid [TDPA]

The core reaction using TDPA as the phosphonate ligand was demonstratedas a control reaction. This reaction proceeded with an initial emissionreading at 1 minute of ˜490 nm and progressing to a final emissionreading of ˜544 nm at 14 minutes. The full width at half maximumintensity (FWHM) never got above 28 nm. The final “growth solution” ofthe cores was yellow/light orange in appearance by eye. The aliquotedsamples of this reaction remained dispersed and clear solutions inhexane.

Core Reaction with 11-methoxy-11-oxo-undecylphosphonic Acid

The reaction using 11-methoxy-11-oxo-undecylphosphonic acid as thephosphonate ligand proceeded with an initial emission reading at 1minute was ˜560 nm; this was redder than the final emission of thecontrol reaction. The final emission of this reaction was ˜610 nm. TheFWHM of this reaction started at ˜35 nm and steadily got more broadthroughout the reaction for a final FWHM of ˜50 nm.

The aliquoted samples were not soluble in hexane, and became almostinstantly flocculated and settled to the bottom of the vials withinminutes.

Core Reaction with 6-ethoxy-6-oxohexylphosphonic Acid

The core reaction using 6-ethoxy-6-oxohexylphosphonic acid as thephosphonate ligand had an initial emission reading at 1 minute of ˜560nm and a final emission reading of ˜606 nm. The FWHM of this reactionstarted out at 1 minute at ˜43 nm and narrowed to a final FWHM of ˜40.5nm.

The solubility of the aliquoted samples was observed. The hexane sampleswere immediately cloudy, however the flocculation did not settle to thebottom of the vials. Six of the aliquoted samples were centrifuged andthe resulting clear, colorless supernatants were discarded. The pelletswere soluble in toluene, dichloromethane (CH₂Cl₂), dimethylformamide(DMF), and methanol (MeOH). The pellets were not soluble in water, 50 mMborate buffer at pH=8.3 or hexane.

Particles synthesized in the presence of TDPA are soluble in hexane,toluene, CH₂Cl₂, DMF and hexane. The 6-ethoxy-6-oxohexylphosphonic aciditself is not soluble in hexane, and neither were the resultingparticles from this reaction, suggesting that the ligand was indeedcoating the nanoparticles—a suggestion which was confirmed with infraredand NMR spectroscopy indicating the expected ester functionality. Usinga solvent system of toluene as the solubilizing solvent and hexane as aprecipitating solvent, a pellet can be formed along with a clear,colorless supernatant. The resulting pellet can be re-solubilized intoluene. This resulting toluene solution allowed an absorbance spectrumof these cores to be obtained.

These data suggest that quantum confined nanoparticles have been formedwith 6-ethoxy-6-oxohexylphosphonic acid on the particle surface. Theresulting core particles were taken further into a shell reaction.

Shell Reaction Procedure Using 6-ethoxy-6-oxohexylphosphonic Acid

Core Precipitation

Three (3) mL of growth solution cores using6-ethoxy-6-oxohexylphosphonic acid ligand (prepared according to theprocedure of Example 4) was solubilized into 3 mL toluene in a 250 mLconical bottom centrifuge tube. A total of 135 mL of hexane was added toprecipitate the cores. The tube was centrifuged at 3000 RPM for 5 min.The resulting clear, colorless supernatant was discarded and the pelletwas dispersed into 3 mL of toluene.

Shell Reaction

Into a 25 mL 3 neck flask with 14/20 joints, 1.4 g of TOPO was weighed.To this, 1-1000 mg of 6-ethoxy-6-oxohexylphosphonic acid was added. Astir bar and 1.4 mL of TOP were added to the flask. The flask wasconnected to an inert atmosphere manifold and evacuated thoroughly, thenrefilled with nitrogen. 2.6 mL of the toluene solution of cores wasadded to the flask and the flask was warmed and evacuated to remove thetoluene, then refilled with nitrogen. Approximately 1 mL of a suitablyhigh-boiling amine was added to the flask and the flask was heated to200-350° C. Solutions of suitable cadmium and zinc precursors in TOPwere prepared with a concentration of 0.5 mol metal ion per kg ofsolution. A solution of 10% trimethylsilylsulfide in TOP by weight wasprepared as well. The metal and sulfur precursor solutions were addedslowly over the course of several hours to minimize additionalnanoparticle nucleation. Sufficient shell precursors were added to growa shell of a desired thickness, as can be calculated by one of ordinaryskill in the art. When the desired shell thickness was reached, thereaction was cooled and the core/shell nanoparticles were isolated byconventional means. Aliquots taken during the reaction permittedmonitoring of the progress of the shell reaction. It was observed thatthe emission maximum after heating but before addition of shellprecursors was very similar to that of the initial cores (˜600 nm),suggesting that the bi-functional phosphonate was sufficiently stronglycoordinated to the nanoparticle surface to minimize Ostwald ripening. Ared-shift during shell precursor addition of ˜50 nm was typical of ashell as deposited in a reaction employing TDPA, suggesting that theshell formed as expected. In addition, the nanoparticle solution becamemuch more intensely emissive, as would be expected of successfuldeposition of an insulating shell. Infrared and NMR spectroscopyconfirmed that the functionalized phosphonates were present on thenanoparticles.

Example 11

Conjugates of Active Polymerase and Nanocrystals

Preparing Phi29 Polymerase Conjugated with UDG-ugi-C8 Nanoparticles

His tagged UDG protein (uracil DNA glycosylase) (2.02 mL, 53.4 μM in 30mM Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM EDTA and 1 mM DTT) wasmixed with ugi (uracil-DNA glycosylase inhibitor) (748 μL, 173 μM in 10mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20,0.1 mM EDTA and 50% v/v glycerol) in 1:1.2 molar ratio (His-tagged-UDGto ugi protein), and incubated at 4° C. overnight. The resultingHis-tagged-UDG-ugi protein complex was stored at 4° C. without furtherpurification for future use.

C8 Nanoparticles (100 μL, 5.3 μM in 50 mM borate buffer pH 8 with 1.0 MBetaine which is frozen at −20° C. immediately after synthesis) wasthawed and mixed with the His-tagged-UDG-ugi protein complex (132 μL,40.0 μM in 30 mM Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM EDTA and 1mM DTT) and 389 μL of 100 mM Tris (pH 7.5) buffer with 300 mM NaCl and 1mM DTT in a 1:10 molar ratio (nanoparticle to His-tagged-UDG-ugi). Thesolution was incubated for 1 hour at 4° C. The resultingUDG-ugi-nanoparticles solution was mixed with stock His-tagged HP1-Phi29mutant polymerase (SEQ ID NO:9) (115 μL, 46 μM in 10 mM Tris (pH 7.5)buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20, 0.1 mM EDTA and50% v/v glycerol) in a 1:10 molar ratio (nanoparticle to polymerase).The conjugation solution was incubated overnight at 4° C., centrifugedfor 5 minutes at 16.8K rcf, purified on Ni²⁺-NTA Agarose column using100 mM Tris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT as the eluent.The purified conjugate solution was centrifuged for 5 minutes at 16.8Krcf, transferred into a 10K MWCO dialysis cassette, then dialyzed into50 mM Tris buffer pH7.5 with 150 mM NaCl, 0.2 mM EDTA, 0.5% v/vTween-20, 5 mM DTT and 50% v/v glycerol. The resultingUDG-ugi-nanoparticle-HP1-Phi29 conjugate was assayed to determineconcentration, template extension activity, and active number of Phi29per conjugate and DNA binding by FRET signal detection (see Table 1below).

TABLE 1 Conjugate Activity C8-UDG-ugi-HP1 Phi29 mutant 0.50base/sec/conj Stock HP1 Phi29 mutant 0.10 base/sec/enz

The FRET signals from the mutant Phi29-nanoparticle conjugate binding tooligonucleotide 199 labeled at the 3′ end with ALEXA FLUOR 647(conjugate and C8 dot concentration: 10 nM; AF647-3′-oligo 199concentration: 1000 nM) were compared to non-conjugated C8nanoparticles. The 605/670 ratio is the fluorescence intensity at 605 nmdivided by fluorescence intensity at 670 nm with 450 nm excitation forboth the conjugate and the unconjugated C8 nanoparticles. The low605/670 ratio for the conjugate indicated the conjugate binding to thedye labeled oligo and showing FRET signal.

The active number of polymerases per conjugate for C8-UDG-ugi-HP1 Phi29mutant conjugate are shown in FIGS. 9A, B and C.

Preparing B103 Polymerase Conjugated with UDG-ugi-C8 Nanoparticles

His tagged UDG protein (uracil DNA glycosylase) (2.02 mL, 53.4 μM in 30mM Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM EDTA and 1 mM DTT) wasmixed with ugi (uracil-DNA glycosylase inhibitor) (748 μL, 173 μM in 10mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20,0.1 mM EDTA and 50% v/v glycerol) in 1:1.2 molar ratio (His-tagged-UDGto ugi protein), and incubated at 4° C. overnight. The resultingHis-tagged-UDG-ugi protein complex was stored at 4° C. without furtherpurification for future use.

C8 nanoparticle s (100 μL, 4.5 μM in 50 mM borate buffer pH 8.0 with 1.0M Betaine which were frozen at −20° C. immediately after synthesis) wasthawed and mixed with stock His-tagged HP1-B103 polymerase (SEQ ID NO:4)(40.5 μL, 111 μM in 10 mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mMDTT, 0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v glycerol) and 309 μL of100 mM Tris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT in a 1:10molar ratio (nanoparticle to polymerase). The conjugation solution wasincubated overnight at 4° C. The resulting B103 polymerase-C8nanoparticle conjugate was mixed with the His-tagged-UDG-ugi proteincomplex (112 μL, 40.0 μM in 30 mM Tris buffer (pH 8) with 200 mM NaCl,0.5 mM EDTA and 1 mM DTT) in a 1:10 molar ratio (nanoparticle toHis-tagged-UDG-ugi). The mixture was incubated for 5 hours at 4° C. toprepare the UDG-ugi-nanoparticle s-B103 conjugate. The resultingconjugate solution was centrifuged for 5 minutes at 16.8K rcf, purifiedon Ni²⁺-NTA agarose column using 100 mM Tris (pH 7.5) buffer with 300 mMNaCl and 1 mM DTT as the eluent, centrifuged and transferred into a 10KMWCO dialysis cassette. The conjugate was dialyzed into 50 mM Trisbuffer pH 7.5 with 150 mM NaCl, 0.2 mM EDTA, 0.5% v/v Tween-20, 5 mM DTTand 50% v/v glycerol. The resulting UDG-ugi-nanoparticle-HP1-B103conjugate was assayed to determine concentration, template extensionactivity, active number of Phi29 per conjugate and DNA binding by FRET(see Table 2 below).

TABLE 2 Conjugate Activity C8-HP1 B103-UDG-ugi 1.41 base/sec/conj StockHP1 B103 0.41 base/sec/enz

The FRET signals from the B103-nanoparticle conjugate binding tooligonucleotide 199 labeled at the 3′ end with ALEXA FLUOR 647(conjugate and C8 dot concentration: 10 nM; AF647-3′-oligo 199concentration: 1000 nM) were compared to non-conjugated C8nanoparticles. The 605/670 ratio is the fluorescence intensity at 605 nmdivided by fluorescence intensity at 670 nm with 450 nm excitation forboth the conjugate and the unconjugated C8 nanoparticles. The low605/670 ratio for the conjugate indicated the conjugate binding to thedye labeled oligo and showing FRET signal.

Active number of polymerase per conjugate for C8-HP1 B103-UDG-ugiconjugate are shown in FIGS. 10A, B and C.

Example 12

FRET Detection of Incorporated Nucleotides Using Polymerase-DyeConjugates

Preparing PEG-Biotin Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and biotin-PEG to produce a low densitybiotin surface with a PEG coating to prevent non-specific background ofproteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of containing approximately 2 μl of fluid.

Attaching Biotinylated DNA to Low Density PEG-Biotin Surfaces:

Streptavidin protein was diluted to 200 pM in Incubation Buffer (50 mMNaCl; 50 mM Tris-Cl pH=7.5; 0.5% BSA). Diluted streptavidin was flowedinto fluidic chamber and streptavidin was incubated for 10 minutes.Chambers were washed 1× with 1 ml Incubation Buffer. Biotinylated-DNAtemplates were diluted to 200 pM in Incubation Buffer and allowed tobind for 5 minutes. Surfaces were washed 1× with 1 ml Incubation Buffer.

SA-Polymerase Preparation:

Streptavidin was labeled with Cy3 (Life Technologies). Streptavidin-Cy3was mixed with a biotinylated mutant Phi29 (b-Phi29)(SEQ ID NO:12) at a1:1 ratio of SA-protein:biotinylated-Phi29 in 1×PBS.

SA-Cy3-b-Phi29 Binding to Templates:

SA-Cy3-b-Phi29 was diluted to 1 nM in binding buffer (50 mM Tris-Cl;pH=7.5; 0.3% BSA; 100 mM NaCl). Conjugates were flowed into fluidicchamber which were previously loaded with DNA templates on the surface.Surfaces were incubated for 5 minutes with 1 nM SA-Cy3-b-Phi29. Surfaceswere washed with 1×1 ml Incubation Buffer.

Fluorescence Imaging:

The Olympus microscope body was outfitted with a TIRF objective lens(100×; 1.45 NA). The excitation light passes through an excitationfilter (EX FT-543/22), and dichroic mirror (DM-532) and the sample isepi-illuminated (Coherent) using TIR at typically 100 W/cm². Uponexcitation, resulting epifluorescence emission passes an emission filter(EM FT-540LP) and the resulting emission is split into three paths(triview) using 2 dichroic mirrors and the appropriate bandpass filtersfor the dye sets of choice. The emission was imaged on a CCD camera.Images were collected at a frame rate of approximately 30 ms. Imagesdepict single DNA strands complexed with single SA-Cy3-b-Phi29conjugates (donor molecules in this example) and FRET signals fromacceptor species (hexaphosphate nucleotides labeled with ALEXA FLUORE647, 676 or 680) bound in the enzyme active site.

Nucleotide Polymerization with SA-DonorDye-Phi29 or B103 Conjugates:

Homopolymer Template Sequence:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer. (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 mM 1,2 phenylenediamine; 100mM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTrolox). Nucleotide mix was flowed into channel with SA-Cy3-b-Phi29 orSA-Cy3-b-B103 (SEQ ID NO:5) bound to DNA template and images wererecorded for approximately 2 minutes at approximately 20 ms frame rates.

As one example, the DNA template sequence extends with the followingsequence (G)₁₅ (A)₁₅(G)₁₅ (A)₁₅ (SEQ ID NO:39). Using 200 nMhexaphosphate-nucleotide 647-dGTP and 200 nM hexaphosphate-nucleotide676-dATP, patterns were identified with spectral signatures for 647 dyeemission (G signal) preceding spectral signatures for 676 dye emission(e.g. the A signal) which resulted from fluorescence resonance energytransfer (FRET) from the donor molecule SA-Cy3-b-Phi29, orSA-Cy3-b-B103.

Random Template Sequence:

Hexa-phosphate nucleotides which were dye-labeled at the terminalphosphate group were diluted to 200 nM in extension buffer. (50 mM MOPSpH=7.1; 75 mM potassium acetate (pH=7.0); 0.3% BSA; 1 mM MnCl₂; 300 nMprocatuate dioxygenase; 4 mM 3,4 dihydroxyl benzoic acid; 1 mM2-nitrobenzoic acid; 400 mM 1,2 phenylenediamine; 100 mM ferrocenemonocarboxylic acid; 0.02% cyclooctratetraene; 6 mM Trolox). Nucleotidemix was flowed into channel with SA-Cy3-b-Phi29 bound to DNA templateand images were recorded for approximately 2 minutes at approximately 20ms frame rates. The DNA template sequence extended with the followingsequence:

Random oligonucleotide: (SEQ ID NO: 40)5′-TTGAACGGATGAGGACCAGACACCACTTGAACGGATGAGGAAAAAA AAAATCA-3′.

Using 200 nM hexaphosphate-647-dGTP and 200 nM hexaphosphate-676-dATP, 2μM dCTP and 2 μM dTTP, patterns were identified with spectral signaturesfor 647 dye emission (G signal) and 676 dye emission (A signal) whichresulted from fluorescence resonance energy transfer (FRET) usingSA-Cy3-b-Phi29 or SA-Cy3-b-B103 as the donor molecule, respectively.

Analysis of Homopolymer Sequencing Results.

The resulting pattern sequencing data acquired using the methodologiesdescribed in Example 12 herein was processed. The subsequent detectedFRET events were filtered and sequences were aligned.

In one exemplary experiment, 200 nM hexaphosphate-647-dGTP and 200 nMhexaphosphate-676-dATP was used along with SA-Cy3-b-Phi29 as the donormolecule. The alignment algorithm found 55 molecules in the field ofview, which clearly demonstrated the completion of at least 30 basepairs of the full 60 base pair sequence. As a result, the number ofevents detected for the first 15 G insertions was approximately 15 andthe number of events for the subsequent 15 A insertions wasapproximately 15.

In another exemplary experiment, 200 nM hexaphosphate-647-dGTP and 200nM hexaphosphate-676-dATP was used along with SA-Cy3-b-B103 as the donormolecule.

Analysis of Random Sequencing Results.

Resulting pattern sequencing data acquired using the methodologiesdescribed in Example 13 herein was processed. The subsequent detectedFRET events were filtered and sequences were aligned. Results arerepresented by the aligning the sequence, whereby light gray blocksrepresents the G insertion signals and dark gray indicates A insertionsignals. The alignment algorithm found 95 molecules in the field ofview, which demonstrated the matching of detected events with the actualsequence.

Enzyme Kinetics and Extension Speeds for Polymerase Conjugates:

Using the conditions and template described above in the homopolymersequence example, various SA-Cy3-labeled mutant polymerase conjugateswere tested to determine on-chip single molecule kinetics and extensionspeeds.

Distributions of the start times were shown for all of the events fromall of the molecules which were successfully aligned with the algorithmfrom their respective blocks, whereby a block refers to 1 of the 4homopolymer stretches. This analysis demonstrates the nearly uniformextension speed of the population of SA-Cy3-polymerase conjugates. Inaddition, the distributions for event duration from each of the blocksfor all of molecules with correct sequence alignment was also determinedand found to provide strong correlation with stopped-flow experiments.

Example 13

Analysis of Fluorescence Data to Extrapolate Sequence Information

To convert the observed fluorescence emissions detected during thenucleotide incorporation reaction into nucleotide sequence information,the raw data comprising a movie of observed emissions was firstprocessed by using a Hidden Markov Model (HMM)-based algorithm to detectand identify FRET events. The subsequent detected FRET events werefiltered and filtered sequences were aligned. Each of these two steps,FRET event detection and sequence analysis, are described in more detailbelow. The HMM-based algorithm was used to analyze the data in Example14 below.

Detection of FRET Events

The analysis underlying FRET event detection is designed to processspatially correlated movie(s) comprising real time sequence fluorescenceemission data, and extract time-series of interest from those data. Amovie typically contains one or more channels where each channelrepresents the same spatial location at different wavelengths. Theanalysis chain begins with the submission of one or more movies to theanalysis machine via a comprehensive user interface. The user interfacerequires the user to input various parameters which describe themovie(s) (e.g. channel regions, dye emission properties). Once this datais submitted the movie(s) are then processed by the image analysissoftware where a sliding window of N frames propagates through the moviecalculating a temporal local average of the frames within the window. Ateach position of the window in the movie, the local average image isthen further processed and enhanced using well known image processingalgorithms and a record of the maximum projection of all the localaverage images is recorded to produce a global image of the movie. Thisglobal image is the input into a spot identification algorithm whichproduces a set of spots identified by a unique spot identification, itsx and y location and its corresponding channel Each set of spots for agiven channel is then registered to the set of spots in every otherchannel. In this way a set of spot tuples is constructed. If a detectedspot in one channel does not have a corresponding detected spot inanother channel, then the position of the undetected spot using thetransformation between the two channels and the location of the detectedspot is inferred. Once a complete set of spot tuples is constructed themovie is iterated over and at each frame the amplitude of each spot iscalculated and appended to the appropriate time-series.

The collection of time-series from a spot tuple consists of time-seriesfrom donor and corresponding acceptor channels. This collection iscalled a Vector Time-Series (VTS). The FRET detection process startswith a data segmentation step using a Markov Chain Monte-Carlo (MCMC)algorithm. Each segment of VTS is modeled by a multivariate Gaussianmodel, with each of the channel modeled by a mean and a standarddeviation. This model establishes a baseline for each channel, fromwhich quantities such as “Donor Down” and “Acceptor Up” can becalculated. A Hidden Markov Model (HMM) was used to model the observeddata. The underlying states consist of a null state, a blink state and anumber of FRET states (one for each acceptor channel). Each state hasits emission probability, which reflects the state's correspondingphysical concept. FRET states are characterized by significant “donordown” and “acceptor up” signals. Blink state is characterized bysignificant “donor down” with no “acceptor up”. Null state ischaracterized by no “donor down” and no “acceptor up”. Given theobserved VTS signal, the emission matrix, and a state transitionprobability matrix, the most probable state path can be computed usingthe Viterbi algorithm. This state path assigns each of the frames to astate. Temporally neighboring FRET frames are grouped into FRET events.For each of the detected FRET events, a list of event features arecalculated, including event duration, signal average, signal to noiseratio, FRET efficiency, probability of event, color calling and otherfeatures. This list of events and corresponding features are stored in afile.

The final stage of the automated analysis generates a report summarizingthe results in the form of a web page containing summary image,statistics of the spots and FRET detection, together with line intensityplots and base call plots. See for example, Watkins et al., “Detectionof Intensity Change Points in Time-Resolved Single-MoleculeMeasurements” J. Phys. Chem. B., 109(1):617-628 (2005).

Using the above process, the movie data obtained from the sequencingreactions was analyzed to detect and identify FRET events according tothe process described above. The FRET events were then processed toidentify sequences as described below.

Sequence Analysis

Beginning with the set of detected Forster resonance energy transfer(FRET) events, a data overview was constructed in the form of a colorimage interpreted as a sequencing plot. To generate the plot, theoriginal FRET event data was pre-processed using a set of filtersconstructed by a priori knowledge of the sequence. For each reactionsite (each molecule) an ordered sequence of FRET events was constructed.The base call letters for each FRET event (e.g. “A”, “C”, “G” or “T”)were concatenated to form a sequence ASCII string. The order of lettersin the string reflects the temporal relationship of the events. Giventhat the expected sequence was known a priori, a regular expression wasthen constructed which represented the full or partial expected sequenceor sequence pattern. Matching against the regular expression (expectedsequence) was then computed for each sequence in the set and the startand stop indices of the match were recorded. A color plot image was thenconstructed where each row corresponds to a sequence in the set. Theplot image was padded to accommodate sequences of different lengths. Acolor map of 2*N+1 colors was constructed, where N denotes the number ofpossible base calls in each sequence (N=2 for the plot of this Example).N colors were assigned to the base characters which fell within thepattern, N colors were assigned to the base characters which did notfall within the pattern (muted color), and finally a color was assignedto the padding (background) of the image. The rows of the image werethen sorted according to the number of base calls in the first part ofthe sequence pattern. The rows of the image were also aligned such thatthe start of the expected sequence is in the same column for all rows ofthe plot.

Example 14

Nucleotide Incorporation with B103 Polymerase-Nanoparticle Conjugates:

Template oligonucleotide: (SEQ ID NO: 41) 5′ TTTTGA TT CCCCC TT CCCCC GACA CGG AGG TTC TAT CAT CGT CAT CGT CAT CGT CAT CG-Biotin TEG-T-3′Primer oligonucleotide: (SEQ ID NO: 42)5′-CGATGACGATGACGATGACGATGATAGAACCTCCGTGTC-3′ (SEQ ID NO: 55) Theexpected sequence is: GGGGGAAGGGGGAA

A template/primer duplex was formed by mixing 1 μL template (100 μM) and0.5 μL of primer (250 μM) in 48.5 μL of buffer composed of 50 mM Tris pH7.5, 50 mM NaCl and 10 mM MgCl₂. The mixture was incubated at 98° C. for2 minutes. The mixture was incubated for 30 minutes at room temperature.

Polymerase/Nanoparticle stocks: C8 nanoparticles-UDG-ugi-HP1-Phi29mutant A, 0.17 μM stock concentration. C8 nanoparticles-44-UDG-UGi-Phi29 mutant B; 0.38 μM stock concentration. C8nanoparticles-38-L-B103-UDG-Ugi (SEQ ID NO:4); 0.38 μM stockconcentration.

Functionalized nanoparticles were diluted to 2 nM in 100 μL of buffercomposed of 50 mM MOPS pH 6.8, 200 mM NaCl, and 0.3% BSA.

The chip was prepared as follows: dd H₂O (1 mL/lane). Wash buffer wash(0.2 mL/lane). Inject lane w/ 5 nM SA and incubate for ˜10 minutes. Washbuffer wash (0.4 mL/lane). Inject 200 pM 355/366 duplex for and incubatefor ˜5 minutes. Wash buffer wash (0.4 mL/lane). Polymerase bindingbuffer wash (0.2 mL/lane). Mount on scope. Inject 2-5 nM of conjugateand incubate until desired density is reached (<700 spots/FOV).Polymerase binding buffer wash (0.2 mL/lane). Inject 1× extension mix(w/o nucleotides) ˜3 min (0.1 mL/lane). Inject 1× extension mix withnucleotides (0.1 mL/lane).

The template/primer duplex (200 pM) was immobilized on biotin-embedded,PEG-coated glass slides purchased from Microsurfaces, Inc. (Bio-01 PEG,Austin, Tex.) using 5 nM streptavidin. The functionalized nanoparticle(2 nM) were conjugated to the surface immobilized duplexes. Theconjugates were washed with 100 μL of buffer composed of 50 mM MOPS pH6.8, 50 mM potassium-OAc, 2 mM MnCl₂, 0.3% BSA, 100 U/mL glucoseoxidase, 10 U/μL Katalase, 10 mM Trolox (dissolved in 24 mM MOPS pH6.8), 0.1% Tween-20, 2 mM (Asp)₄ (SEQ ID NO: 67), and 0.5% glucose.

The extension reaction was initiated by injecting 100 μL of buffercomposed of 50 mM MOPS pH 6.8, 50 mM potassium-OAc, 2 mM MnCl₂, 0.3%BSA, 100 U/mL glucose oxidase, 10 U/μL katalase, 10 mM Trolox, 0.1%Tween-20, 2 mM (Asp)₄ (SEQ ID NO: 67), 0.5% glucose, 0.2 μMAF647-terminal phosphate labeled dG6P, and 0.2 μM AF680-terminalphosphate labeled dA6P. Laser excitation: 405 nm, ˜19 W/cm2, 16 ms.

As described in Example 13 above, the fluorescent signals emitted by thenucleotide incorporation reaction were captured in a movie, and theimages were processed using the Hidden Markov Model (HMM).

Example 15

Nucleotide Incorporation with B103 Polymerase-Nanoparticle Conjugates:

Template 404 sequence: (SEQ ID NO: 43)5′-TGATTTTTTTTTTCCTCATCCGTTCAAGTGGTGTCTGG TCCTCATCCGTTCAAGACA CGG AGGTTC TAT CAT CGT CAT CGT CAT-biotin TEG-T-3′ Primer 317 sequence: (SEQ IDNO: 44) 5′ TGA TAG AAC CTC CGT GT 3′Duplex Template/Primer Preparation:

The template oligonucleotide, at 100 nanomolar concentration, and theprimer oligonucleotide, at 1 micromolar concentration, were heated to98° C. in annealing buffer (50 mM Tris, pH 7.5, 50 mM NaCl) for 5minutes and allowed to cool to room temperature.

Flow Chamber Preparation

PEG/PEG-biotin coated cover slips (MicroSurfaces, Inc., Minneapolis,Minn.) were assembled into 9-lane reaction chambers with laser-cut 3Madhesive and custom fabricated plastic superstructures with inlet/outletports for fluid addition. The surface was wetted by flowing 1 milliliterof Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) containing0.1% Tween-20 and 0.5% bovine serum albumin (Sigma, Cat.# A8577)(TBST-B) into each chamber and incubating at room temperature for 5minutes. The surface was coated with streptavidin by flowing 100microliters of 5 nM streptavidin, (Zymed, Cat #43-4302) diluted inTBST-B, and incubating for 30 minutes at room temperature. The laneswere washed with 1 milliliter of TBST-B. The duplex template/primer wasdiluted to 5 pM in TBST-B and 100 microliters was flowed into thereaction chamber and incubated 30 minutes at room temperature. The laneswere washed with 1 milliliter of TBSB.

Nucleotide Incorporation Reaction

Polymerase-nanoparticle conjugates are bound to the templates in theflow chamber for one minute in binding buffer (50 mM MOPS, pH 7.0, 2 mM4-Aspartate (SEQ ID NO: 67), 50 mM Potassium Acetate, 0.3% BSA, 2 mMMnCl₂, 0.1% tween-20, 0.5 mg/ml glucose Oxidase, 10 U/ul Katalase, 10 mMTrolox (in ethanol), 0.5% glucose) at a concentration of 10 nM. Thepolymerase was exo-minus Phi29 (SEQ ID NO:9). Excess unbound conjugateis washed off with 200 microliters of TBST-B. Image acquisition isinitiated and 100 microliters of extension buffer (binding buffer with200 nanomolar each 680-dG6P and 647-dA6P and 1 micromolar each dTTP anddCTP) was flowed through the chamber. Images are acquired for 90 secondsand exposure time was 16 ms. 405 nanometer laser power density was 20W/cm².

As described in Example 13 above, the fluorescent signals emitted by thenucleotide incorporation reaction were captured in a movie, and theimages were processed using the Hidden Markov Model (HMM).

Example 16

Nucleotide Incorporation with B103-Fluorescent Dye Conjugates

Preparing NHS-Ester Surfaces:

Glass coverslips surfaces were plasma cleaned and treated with a mixtureof poly-ethyleneglycol (PEG) and NHS-ester to produce a low densityNHS-ester surface with a PEG coating to prevent non-specific backgroundof proteins and macromolecules.

Fluidic Chamber Assembly:

Fluidic cassettes were assembled with glass coverslips to create fluidicchambers capable of carrying approximately 2 μl of fluid.

Attaching Amine Terminated Hairpin DNA to Low Density NHS-EsterSurfaces:

Target DNA hairpin sequence: (SEQ ID NOS 45 and 65, respectively)5′-TTTTTTTTACCCCCGGGTGACAGGTTXTTCCTGTCACCC-3′where “X” is an amine group.

The target DNA was diluted to 500 nM in 1 M NaHCO₃. The diluted targetmolecules were flowed into the fluidic chamber and incubated for 1 hour.Chambers were washed 1× with 1 ml deactivating buffer (ethanolamine).Surfaces were washed 1× with 1 ml incubation buffer (50 mM Tris-Cl,pH=7.5; 50 mM NaCl; 0.3% BSA).

SA-Polymerase Conjugate Preparation:

Streptavidin was labeled with Cy3. Streptavidin-Cy3 was mixed withbiotinylated-B103 (b-B103-exo minus)(SEQ ID NO:5) at a 1:1 ratio ofSA-protein: biotinylated-B103 in 1×PBS.

SA-Cy3-b-B103 Binding to Templates:

The SA-Cy3-b-B103 conjugates were diluted to 1 nM in binding buffer (50mM Tris-Cl; pH=7.5; 0.3% BSA; 100 mM NaCl). The conjugates were flowedinto the fluidic chamber which were previously loaded with DNA templateson the surface. Surfaces were incubated for 5 minutes with 1 nMSA-Cy3-b-B103. Surfaces were washed with 1×1 ml incubation buffer.

Fluorescence Imaging:

The microscope body was purchased from Olympus and was outfitted with aTIRF objective lens (100×; 1.45 NA). The excitation light passes throughan excitation filter (EX FT-543/22), and dichroic mirror (DM-532) andthe sample was epi-illuminated (Coherent) using TIR at typically 100W/cm². Upon excitation, the resulting epifluorescence emission passedthrough an emission filter (EM FT-540LP) and the resulting emission wassplit into three paths (tri-view) using 2 dichroic mirrors and theappropriate bandpass filters for the dye sets of choice. Using thisfilter combination, we were able to spectrally resolve 1 donor dye and 3acceptor dyes in 3 detection channels.

In separate experiments, 1 donor dye and 4 different acceptor dyes couldbe resolved in 4 detection channels. The optical detection scheme was asfollows: DC1=635, F1 640LP; DC2=675, F2=688/31; DC3=705, F3=700 LP. Thedonor dye used in this case was CY3 and the 4 acceptor dyes are asfollows DY634, AF647, AF676, AF700

The emissions resulting in each experiment were imaged on a CCD camera.Images were collected at a frame rate of approximately 20 ms.

Three-Color Nucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with SA-Cy3-b-B103 boundto DNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates. In this example, the synthesized strandis expected to have the following sequence: (G)₅T(A)₈ (SEQ ID NO: 66).Terminal phosphate-labeled nucleotides and 125 nM cold dC6P were usedfor the nucleotide incorporation reaction. The labeled nucleotidesincluded 125 nM 647-dT6P, 125 nM 676-dG6P, 125 nM 700-dA6P. The spectralsignatures for the ALEXA FLUOR-676 G signal, AF-647 T signal, and AF-700A signal were identified that resulted from fluorescence resonanceenergy transfer (FRET) from the Cy3 donor molecule, and corresponded tothe correct insertion sequence pattern.

Analysis of Three-Color Sequencing Results

Resulting pattern sequencing data was processed using an alignmentalgorithm. The alignment algorithm found 100 molecules in the field ofview, which demonstrated completion of the full 14-nucleotide sequence((G)₅T(A)₈ (SEQ ID NO: 66), which represented approximately 20% of thetotal single molecule donor population. The consensus sequence wasdetermined using an HMM alignment algorithm (e.g., see Example 14). Byplotting the accuracy definition (measured as a percentage value)against the HMM score (X axis), a linear relationship was detected.Various measurements of accuracy can be devised that can be suitable forsuch analysis. In one exemplary experiment, the accuracy was estimatedaccording to the following equation:

${\alpha\left( {T,A} \right)} = \frac{\beta - \delta - \eta + \lambda}{2\lambda}$

The measurement of accuracy in the above equation is intended to providesome measure of similarity between some given template, T, and somealignment, A, of an observed sequence O. It should be noted thatalphabet of T, A, and O are identical. The length of T is denoted by thenumber of deletions in the alignment A by δ, the number of insertions inthe alignment by η, and the number of matches in the alignment by β.Equation (1) is normalized by λ such that a an accuracy of 1 indicates atotal agreement, and an accuracy of 0 indicates no agreement between Tand A. The above definition of accuracy is provided as an example onlyand is in no way intended to limit the disclosure to any particulartheory or definition of accuracy; alternative definitions of accuracyare also possible and it may be suitable to use such alternativedefinitions in some contexts.

The accuracy in this system using an HMM alignment threshold of 0 wasestimated to be approximately 80%.

Four-Color Nucleotide Incorporation Reaction:

Template molecule: (SEQ ID NO: 46) TTTTTCCCCGACGATGCCTCCCC g ACA Cgg AggTTC TAT CAT CgT CAT CgT CAT CgT CAT Cg-Biotin TEG-T-3 Primer for thetemplate: (SEQ ID NO: 47) 5′ TGA TAG AAC CTC CGT GTC 3′

In this example, the synthesized strand is expected to have thefollowing sequence:

(SEQ ID NO: 48) GGGGAGGCATCGTCGGGAAAANucleotide Incorporation Reaction:

Hexa-phosphate dye-labeled nucleotides were diluted to 200 nM inextension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0);0.3% BSA; 1 mM MnCl₂; 300 nM procatuate dioxygenase; 4 mM 3,4 dihydroxylbenzoic acid; 1 mM 2-nitrobenzoic acid; 400 μM 1,2 phenylenediamine; 100μM ferrocene monocarboxylic acid; 0.02% cyclooctratetraene; 6 mMTROLOX). Nucleotide mix was flowed into channel with SA-Cy3-b-B103 boundto DNA template and images are recorded for approximately 2 minutes atapproximately 20 ms frame rates.

The terminal phosphate-labeled nucleotides used for the nucleotideincorporation reaction included 125 nM DY634-dA6P, 125 nM AF647-dT6P,125 nM AF676-dG6P, 125 nM AF700-dC6P. The spectral signatures for theDY-634 A signal, and the ALEXA FLUOR G, T and C signals (AF-676 Gsignal, AF-647 T signal, and AF-700 C signal) were identified thatresulted from fluorescence resonance energy transfer (FRET) from the Cy3donor molecule, and corresponded to the correct insertion sequencepattern. 4-color sequence alignment was obtained by visual inspection.

The observed FRET event durations for various SA-Cy3-b-B103 conjugates,the event count distributions, and the observed extension speeds ofvarious SA-Cy3-b-B103 conjugates were calculated.

Example 17

Nucleotide Incorporation Reactions

Template oligonucleotide: (SEQ ID NO: 49) 5′-TTTTTCCCCGCGTAACTCTTTACCCCg ACA Cgg Agg TTC TAT CA-3′ Primer oligonucleotide: (SEQ ID NO: 50)5′-TGATAGAACCTCCGTGTC-3′

A duplex was formed by mixing 1 μL template (100 μM) and 4 μL of primer(50 μM) in 21 μL of buffer composed of 50 mM Tris pH 7.5, 50 mM NaCl and10 mM MgCl₂. The mixture was incubated at 98° C. for 2 minutes. Themixture was incubated for 30 minutes at room temperature.

Dye-polymerase conjugate: Cy3 (9.3)-SAV-biotin-(HBP1)-(B103H370R)-(exo)(SEQ ID NO:3), 0.60 μM stock concentration. The polymerase isHBP1-B103(exo-) conjugated to Cy3 via streptavidin/biotin.

The polymerase conjugate was diluted to 0.75 nM in 100 μL of buffercomposed of 50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3% BSA. The duplex(200 pM) was immobilized on biotin-embedded, PEG-coated glass slidespurchased from Microsurfaces, Inc. (Bio-01 PEG, Austin, Tex.) using 0.3nM streptavidin. The dye-polymerase conjugate (0.75 nM) was conjugatedto the surface-immobilized duplexes. The conjugate was washed with 100μL of buffer composed of 50 mM MOPS pH 7.1, 50 mM KOAc pH 6.85, 2 mMTrolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9U/μL katalase and 0.4% glucose.

The extension reactions were initiated by injecting 100 μL of one of thefollowing buffers composed of 50 mM MOPS pH 7.1, 50 mM KOAc pH 6.85, 2mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase,19.9 U/μL katalase, 0.4% glucose, 0.5 mM MnCl₂ and one of the threefollowing nucleotides combinations: (1) 120 nM 647G (AF647-terminalphosphate labeled dG6P), 150 nM 676A (AF676-terminal phosphate labeleddA6P), 3 μM dTTP; (2) 24 nM 647G (AF647-terminal phosphate labeleddG6P), 24 nM 676A (AF676-terminal phosphate labeled dA6P), 24 nM dTTP;and (3) 30 nM 647T (AF647-terminal phosphate labeled dT6P), 24 nM 676G(AF676-terminal phosphate labeled dG6P), 24 nM 700A (AF700-terminalphosphate labeled dA6P).

The HMM-based algorithm described in Example 14 was used to analyze thedata.

Example 18

Nucleotide Incorporation Reactions

Target oligonucleotide: (SEQ ID NO: 49) 5′-TTTTTCCCCGCGTAACTCTTTACCCC gACA Cgg Agg TTC TAT CA-3′ Primer oligonucleotide: (SEQ ID NO: 50)5′-TGATAGAACCTCCGTGTC-3′

A DNA duplex was formed by mixing 1 μL template (100 μM) and 4 μL ofprimer (50 μM) in 21 μL of buffer (50 mM Tris pH 7.5, 50 mM NaCl and 10mM MgCl₂) and incubated at 98° C. for 2 minutes. And the mixture wasincubated for 30 minutes at room temperature.

A dye-conjugated, exo minus, B103 mutant polymerase (B103-H370R) (SEQ IDNO:3) (60 μM stock concentration) was used. The polymerase-dye conjugatewas diluted to 0.75 nM in 100 μL of buffer (50 mM MOPS pH 7.03, 100 mMNaCl, and 0.3% BSA).

The DNA duplex (200 pM) was immobilized on biotin-embedded, PEG-coatedglass slides purchased from Microsurfaces, Inc. (Bio-01 PEG, Austin,Tex.) using 0.3 nM streptavidin. The polymerase-dye conjugate (0.75 nM)was reacted with the surface-immobilized DNA duplexes. The DNAduplex/polymerase complex was washed with 100 μL of buffer (50 mM ACESpH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02% cyclo-octatetraene (COT),200 U/mL glucose oxidase, 19.9 U/μL katalase and 0.4% glucose).

The extension reactions were initiated by injecting 100 μL of one of thefollowing buffers composed of 50 mM MOPS pH 7.1, 50 mM KOAc pH 6.85, 2mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase,19.9 U/μL katalase, 0.4% glucose, 0.5 mM MnCl₂ following nucleotidescombinations: 30 nM 647T (AF647-terminal phosphate labeled dT6P), 24 nM676G (AF676-terminal phosphate labeled dG6P), 24 nM 700A (AF700-terminalphosphate labeled dA6P).

The HMM-based algorithm described in Example 14 was used to analyze thedata.

Example 19

Nucleotide Incorporation with Polymerase-Tripod Nanoparticle Conjugates

Preparing Tripod Nanoparticle-His-B103-H370R(exo-) Polymerase Conjugates

Tripod Nanocrystals (50 μL, 2.7 μM in 50 mM borate buffer pH 8.0) weremixed with stock His-tagged HP1-B103 H370R exo-polymerase(SEQ ID NO:3)(25 μL, 16 μM in 10 mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mM DTT,0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v glycerol) and 40 μL of 100 mMTris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT in a 1:3 molar ratio(nanocrystal to polymerase). The conjugation solution was incubatedovernight at 4° C. The resulting conjugate solution was centrifuged for5 minutes at 16.8K rcf, purified on Ni²⁺-NTA Agarose column using 100 mMTris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT as the eluent,centrifuged and transferred into a 10K MWCO dialysis cassette. Theconjugate was dialyzed into 50 mM Tris buffer pH7.5 with 150 mM NaCl,0.2 mM EDTA, 0.5% v/v Tween-20, 5 mM DTT and 50% v/v glycerol. Theresulting Tripod-nanocrystal-HP1-B103 H370R exo-conjugate was assayed todetermine concentration, template extension activity, active number ofPhi29 per conjugate and DNA binding by FRET. In a DNA extension assay,the Tripod-nanoparticle-HP1-B103-H370R (exo-) conjugates exhibited 0.35base/sec/conjugate, and the stock HP1-B104-H370R (exo-) polymeraseexhibited 0.29 base/sec/enzyme.

Nucleotide Incorporation Using the Conjugates

To a 50 μL solution of 10 μM template DNA:Biotin-5′-TTTTTCCCCGCGTAACTCTTTACCCCgACACggAggTTCTATCA-3′-amine) (SEQ IDNO:49), was mixed in 50 μL of 50 μM primer DNA (5′-TGATAGAACCTCCGTGTC-3′(SEQ ID NO:50)). The mixture was heated at 98° C. for 1 minute andchilled on ice. The annealed template/primer was diluted to 200 pM using500 mM borate buffer (pH 8.2), and injected into lanes of a microfluidicdevice with coverslip containing NHS ester reactive groups on thesurface, incubated at room temperature for 10 minutes. The coverslipsurface was deactivated by incubating with 50 mM glycine in 500 mMborate buffer (pH 8.2) for 10 minutes, washed with 50 mM Tris buffer (pH7.5) with 50 mM NaCl, 0.5% BSA and 0.05% Tween-20.

The microfluidic device was secured on a TIRF (total internal reflectionfluorescence) microscope. The TIRF microscope was setup on TIRF modewith power density at ˜15 W/cm² for the 405 nm excitation laser.Nanoparticle-polymerase conjugate solution (10 nM in GO-Cat OSS buffersystem, 50 mM MOPS buffer pH 7.2 with 50 mM KOAc, 0.1% Tween-20, 10 mMTrolox, 0.3% BSA, 0.5 mg/mL glucose oxidase, 10 unit/uL catalase, 2 mMtetra-aspartic acid (SEQ ID NO: 67) and 0.5% freshly added glucose) wasinjected into a lane of the microfluidic, incubated at room temperaturefor ˜1 minute, then washed with the GO-Cat OSS buffer system (50 mM MOPSbuffer pH 7.2 with 50 mM KOAc, 0.1% Tween-20, 10 mM Trolox, 0.3% BSA,0.5 mg/mL glucose oxidase, 10 unit/uL catalase, 2 mM tetra-aspartic acid(SEQ ID NO: 67) and 0.5% freshly added glucose). The successivenucleotide incorporation was captured on a movie, which was recorded for100 seconds at 30 ms per frame rate on a new FOV (field of view) wheninjecting into the lane of a primer extension reaction mixture (e.g. 150nM dG6P-C6-AF647, 150 nM dA6P-C6-AF680, 1000 nM dTTP, 1000 nM dCTP and0.5 mM MnCl₂ in GO-Cat OSS buffer system containing freshly added 0.5%glucose. The movie was analyzed to identify the incorporated nucleotidesusing time series extraction and base calling software.

Example 20

Reagent Exchange Reactions

Target molecule: (SEQ ID NO: 51) 5′TTTTGA TTTTTTTTTTTT CCCCCCCCCCCCTTTTTTTTTTTT CC CCCCCCCCCC g ACA Cgg Agg TTC TAT CAT CgT CAT CgT CAT CgTCAT Cg-amine-3′ Primer molecule A for cycle 1: (SEQ ID NO: 52) 5′ TGATAG AAC CTC CGT GTC 3′ Primer molecule B for cycle 2: (SEQ ID NO: 53)5′ TGA TAG AAC CTC YGT GTC 3′ (Y = amino modifier C6, C is base, labeledwith AF647)1×TBST/BSA Wash buffer: 50 mM Tris pH 7.5; 50 mM NaCl; 0.05% Tween-20;0.5% BSA.1× Polymerase binding buffer: 50 mM MOPS pH 6.8; 100 mM NaCl; 0.1% BSA.Pre-Extension mix G.O./Cat OSS:

50 mM MOPS pH 7.2 w/KOH; 50 mM potassium acetate (KOAc) pH 7.0; 2 mMTrolox (dissolved 24 mM MOPS pH 6.8; stored at −20° C.); 0.2%cyclooctratetraene; 100 U/mL glucose oxidase; 10 U/μL Catalase; 0.4%glucose.

Extension mix G.O./Cat OSS:

50 mM MOPS pH 7.2 w/KOH; 50 mM KOAc pH 7.0; 2 mM Trolox (dissolved 24 mMMOPS pH 6.8; stored at −20° C.); 0.2% cyclooctratetraene; 100 U/mLglucose oxidase; 10 U/μL Catalase; 0.4% glucose; 0.6 mM MnCl₂; 100 nMAF647-dG6P; 100 nM AF676-dA6P.

Covalent-DNA Immobilization and Chip Preparation:

Coverslips from MicroSurfaces, Inc. were prepared as follows. The lanewas injected with 300 pM the target molecules primed with primer A,dissolved in 500 mM borate pH 8.2 and incubated for approximately 5minutes. The reaction was terminated with 0.1 mL wash (500 mM Borate, pH8.2). NHS deactivation was conducted using Deactivation buffer suppliedby MicroSurfaces, Inc., by injecting 0.08 mL/lane and incubated for morethan 5 minutes. The chip was washed with 1×TBST/BSA (1 mL/lane). Thechip was mounted on the scope.

Cycle 1 Nucleotide Incorporation Reaction:

Polymerase binding buffer wash (0.3 mL/lane) was injected. 2-5 nM of thepolymerase conjugate (Cy3-SA-Phi29 mutant) was injected and incubateduntil desired density was reached (˜900 spots/FOV).

Polymerase binding buffer wash (0.2 mL/lane) was injected. Pre-extensionmix (without nucleotides) ˜3-5 min (0.1 mL/lane) was injected. 1×extension mix with nucleotides (0.1 mL/lane) was injected. For cycle 1,AF647-dG6P and AF676-dA6P terminal phosphate labeled nucleotides wereused. Cycle 1 donors were mapped visually.

Removal of Polymerase and Synthesized Strand:

The polymerase used in cycle 1 was removed using 6.3 M guanidineisothiocyanate, 160 mM Tris pH 9.7, and 2.6 mM EGTA. The synthesizedstrand was removed using 25% Formamide, 50 mM NaOH.

Cycle 2 Exchanged Polymerase and Primer:

For cycle 2, 500 nM of fresh, AF647-labeled primer B was added in1×TBST/BSA, and incubated for 5 minutes.

Polymerase binding buffer wash (0.3 mL/lane) was injected. Approximately2-5 nM of the polymerase conjugate (Cy3-SA-Phi29 mutant) was injected.

Polymerase binding buffer wash (0.2 mL/lane) was injected. Pre-extensionmix (without nucleotides) ˜3-5 min (0.1 mL/lane) was injected. 1×extension mix with nucleotides (0.1 mL/lane) was injected. For cycle 2,AF676-dG6P and AF700-dA6P terminal phosphate labeled nucleotides wereused. Cycle 2 donors were mapped visually (same field of view as forcycle 1). Time traces of the fluorescent acceptor signals for cycle 1and 2 were obtained. The number of donors mapped in the same field ofview for cycle 1 and 2 were analyzed.

Example 21

Reagent Exchange Reactions:

In a first cycle, nucleotide incorporation reactions were conducted on atarget nucleic acid molecule with 3 types of nucleotides. The reagentswere exchanged, and a second cycle of nucleotide incorporation reactionswere conducted using 4 types of nucleotides. Accordingly, reagentexchange reactions were performed to continue nucleotide incorporationreactions on the same target nucleic acid molecules.

In this experiment, the nucleotide incorporation reaction proceededtowards the solid surface.

Target hairpin oligonucleotide: (SEQ ID NOS 54 and 65, respectively)5′TTTTTCCCCGACGATGCCTCCCCTTTTTTTTACCCCCGGGTGACAGGT T X TTCCTGTCACCC-3′,where X=amino modifier C6 dT; 5′ biotin.Polymerase conjugated to a Cy3 dye:Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo⁻), 0.60 μM stock concentration.

The polymerase-dye conjugate was diluted to 0.75 nM in 100 μL of buffer(50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3% BSA).

The hairpin oligonucleotide (300 pM) was surface-immobilized onbiotin-embedded, PEG-coated glass slides purchased from Microsurfaces,Inc. (Bio-01 PEG, Austin, Tex.) using 0.5 nM streptavidin. Thepolymerase-dye conjugate (0.75 nM) was reacted with thesurface-immobilized hairpin oligonucleotide (i.e., target nucleic acidmolecule) to produce a polymerase/target complex. The complex was washedwith 100 μL of a pre-extension buffer (50 mM ACES pH 7.1, 50 mM KOAc pH6.85, 2 mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucoseoxidase, 19.9 U/μL katalase and 0.4% glucose).

The first cycle was initiated by injecting 100 μL of extension buffer:50 mM ACES pH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02%cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9 U/μL katalase,0.4% glucose, 0.5 mM MnCl₂ supplemented with the following nucleotidescombinations: 25 nM 647T (AF647-terminal phosphate labeled dT6P), 50 nM676G (AF676-terminal phosphate labeled dG6P), and 25 nM 700A(AF700-terminal phosphate labeled dA6P).

A reagent exchange reaction was performed to remove the polymerase,using 200 μL a solution (4.8 M guanidine isothiocyanate and 200 mM TrispH 9.7). The immobilized hairpin oligonucleotide was washed with 200 μLwash buffer (50 mM Tris pH 7.5, 50 mM NaCl, and 0.3% BSA).

In the second cycle, a fresh supply of the polymerase-dye conjugate(Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo⁻)) was reacted with theimmobilized hairpin oligonucleotide and washed with pre-extension buffer(50 mM ACES pH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02%cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9 U/μL katalaseand 0.4% glucose). The second extension was conducted using theextension buffer supplemented with the nucleotide combination: 25 nM634A (Dy634-terminal phosphate labeled dA6P) 25 nM 647T (AF647-terminalphosphate labeled dT6P), 50 nM 676G (AF676-terminal phosphate labeleddG6P), and 25 nM 700C (AF700-terminal phosphate labeled dC6P).

The full length of the expected extendible sequence is:

(SEQ ID NO: 56) GGGGGTAAAAAAAAGGGGAGGCATCGTCGGGGAAAAA

In the first cycle, the nucleotide incorporation reaction was expectedto produce the underlined sequence shown above. In the second cycle, thenucleotide incorporation reaction was expected to produce thenon-underlined sequence shown above. A time trace of fluorescent signalsfrom cycle 1 and 2 reactions was obtained.

Example 22

Reagent Exchange Reactions:

Reagent exchange reactions were conducted using a polymerase labeledwith a fluorescent donor dye, 4 types of nucleotides each labeled with adifferent fluorescent acceptor dye, and 2 types of non-hydrolyzablenucleotides (unlabeled).

Template oligonucleotide: (SEQ ID NO: 49) 5′-TTTTTCCCCGCGTAACTCTTTACCCCg ACA Cgg Agg TTC TAT CA-3′ Primer oligonucleotide: (SEQ ID NO: 47)5′-TGATAGAACCTCCGTGTC-3′

A duplex was formed by mixing 1 μL template (100 μM) and 4 μL of primer(50 μM) in 21 μL of buffer (50 mM Tris pH 7.5, 50 mM NaCl and 10 mMMgCl₂). The mixture was incubated at 98° C. for 2 minutes. The mixturewas then incubated for 30 minutes at room temperature.

Polymerase conjugated to a Cy3 dye:Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo⁻), 0.60 μM stock concentration.

The polymerase-dye conjugate was diluted to 0.75 nM in 100 μL of buffer(50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3% BSA).

The duplex (300 pM) was immobilized on biotin-embedded, PEG-coated glassslides purchased from Microsurfaces, Inc. (Bio-01 PEG, Austin, Tex.)using 0.5 nM streptavidin.

The nucleotide incorporation and reagent exchange reactions wererepeated 5× on the same target DNA molecules according to the followingprotocol:

The polymerase-dye conjugate (0.75 nM) was reacted with thesurface-immobilized DNA duplex (i.e., target nucleic acid molecule) toproduce a polymerase/target complex. The complex was washed with 100 μLof a pre-extension buffer (50 mM ACES pH 7, 50 mM KOAc pH 6.85, 2 mMTrolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9U/μL katalase and 0.4% glucose).

The extension reactions were initiated by injecting 100 μL of extensionbuffer (50 mM ACES pH 7, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02%cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9 U/μL katalase,0.4% glucose, 0.5 mM MnCl₂, 25 nM 634C (Dy634-terminal phosphate labeleddC6P), 38 nM 647T (AF647-terminal phosphate labeled dT6P), 32 nM 676G(AF676-terminal phosphate labeled dG6P), 38 nM 700A (AF700-terminalphosphate labeled dA6P), 25 nM dApCpp(2′-Deoxy-adenosine-5′-[(α,β)-methyleno]triphosphate, sodium salt) (JenaBioscience, Germany) and 25 nM dUpCpp(2′-Deoxy-uridine-5′-[(α,β)-methyleno]triphosphate, Sodium salt) (JenaBioscience, Germany).

Upon completion of each extension cycle, the polymerase-dye conjugatewas removed with a 200 μL solution (4.8 M guanidine isothiocyanate and200 mM Tris pH 9.7). The immobilized target DNA (now having synthesizedstrands) was washed with 200 μL wash buffer (50 mM Tris pH 7.5, 50 mMNaCl, and 0.3% BSA). The target DNA and synthesized strands wereseparated using a solution (25% v/v formamide and 50 mM NaOH). Thestrand separation reaction was terminated by injecting 200 μL of washbuffer (50 mM Tris pH 7.5, 50 mM NaCl, and 0.3% BSA).

The immobilized target DNA molecules were re-hybridized with primers byinjecting 100 μL of 500 nM the primer dissolved in wash buffer andincubated for 30 minutes. The re-hybridization was terminated byinjecting 200 μL of wash buffer (50 mM Tris pH 7.5, 50 mM NaCl, and 0.3%BSA). A time trace of fluorescent signals from cycle 2 and 3 reactionswere obtained.

Example 23

Nucleotide Incorporation of B103 Polymerase: Stopped Flow Analysis

1) B103 Polymerase: Stopped-Flow Measurements of t_(pol)

Template C sequence: (SEQ ID NO: 57) 5′-CGTTAACCGCCCGCTCCTTTGCAAC-3′Primer sequence: (SEQ ID NO: 58) 5′-GTTGCAAAGGAGCGGGCG-3′

The kinetics of nucleotide incorporation by B103 (exo-) (SEQ ID NO:5)and an H370R mutant (SEQ ID NO:3) DNA polymerases were measured in anApplied Photophysics SX20 stopped-flow spectrometer by monitoringchanges in fluorescence from a duplex Alexa fluor 546 dye-labeled-DNAtemplate following the mixing of the enzyme-DNA complex with dye-labelednucleotides (AF647-C6-dG6P) in the reaction buffer containing 50 mMTris-HCl, pH 7.5, 50 mM NaCl, 4 mM DTT, 0.2% BSA, and 2 mM MnCl₂. Thereactions included 330 nM recombinant DNA polymerase, 100 nMtemplate/primer duplex, and 7 μM labeled nucleotides.

The averaged (5 traces) stopped-flow fluorescence traces (>1.5 ms) werefitted with a double exponential equation (1) to extrapolate the ratesof the nucleotide binding and product release,Fluorescence=A ₁ *e ^(−k) ¹ ^(*t) +A ₂ *e ^(−k) ^(pol) ^(*t)+C  (equation 1)where A₁ and A₂ represent corresponding fluorescence amplitudes, C is anoffset constant, and k1 and kpol are the observed rate constants for thefast and slow phases of the fluorescence transition, respectively. Thedye-labeled nucleotides comprise terminal-phosphate-labeled nucleotideshaving an alkyl linker with a functional amine group attached to thedye. The stopped-flow techniques for measuring t_(pol) (1/k_(pol))followed the techniques described by M P Roettger (2008 Biochemistry47:9718-9727; M. Bakhtina 2009 Biochemistry 48:3197-320).2) B103 Polymerase: Stopped-Flow Measurements of t⁻¹

Template C sequence: (SEQ ID NO: 59) 5′-CAGTAACGG AGT TGG TTG GAC GGCTGC GAG GC-3′ Dideoxy-primer sequence: (SEQ ID NO: 60) 5′-GCC TCG CAGCCG TCC AAC CAA CTC ddC-3′

The rate of the nucleotide dissociation (k⁻1) from the ternary complexof [enzyme⋅DNA⋅nucleotide] was measured in an Applied Photophysics SX20stopped-flow spectrometer by monitoring changes in fluorescence from influorescence from a duplex Alexa fluor 546 dye-labeled-DNA templatefollowing the mixing of the [enzyme⋅DNA⋅labeled nucleotide] ternarycomplex with 50 μM cognate non-labeled deoxynucleoside triphosphate in abuffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM DTT, 0.2%BSA, and 2 mM MnCl₂.

The ternary complexes were prepared using: 330 nM polymerase, 100 nMtemplate/primer duplex, and 7 μM terminal phosphate-labeled nucleotides(AF647-C6-dG6P).

The averaged stopped-flow fluorescence traces (>1.5 msec) were fittedwith a single exponential equation (2) to extrapolate the rate of thenucleotide dissociation (k⁻1) from the [enzyme⋅DNA⋅nucleotide] ternarycomplex.Fluorescence=A ₁ *e ^(−k) ⁻¹ ^(*t) +C  (equation 2)where A₁ represents the corresponding fluorescence amplitude, C is anoffset constant, and k⁻¹ and the observed rate constants for thefluorescence transition. The stopped-flow techniques for measuring t⁻¹(1/k⁻¹) followed the techniques described by M. Bakhtina (2009Biochemistry 48:3197-3208). The results of the stopped-flow experimentsare listed in the table below.

Summary of the t_(pol) and t⁻¹ measurements Polymerase t_(pol) t⁻¹ B103(exo-) 14 16 H370R 17 43 H370Y 15 12 E371R 11 17 E371Y 11 7 K372R 14 12K380R 783 17 D507G 11 13 D507H 7 16 K509Y 10 20 Ph-29 (exo-) 11 27 T373R15 81 T373Y 14 45

What is claimed:
 1. A method for generating an energy transfer signalcomprising the steps of: contacting (i) a polymerase having alterednucleotide incorporation kinetics and linked to an energy transfer donormoiety with (ii) a nucleic acid molecule and with (iii) at least onetype of a nucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide into the nucleic acid molecule therebylocating the polymerase and nucleotide in close proximity with eachother to generate the energy transfer signal; wherein the polymerase isa B103 polymerase comprising SEQ ID NO:1, 2 or
 3. 2. A method forgenerating an energy transfer signal comprising the steps of: contacting(i) a polymerase having altered nucleotide incorporation kinetics andlinked to an energy transfer donor moiety with (ii) a nucleic acidmolecule and with (iii) at least one type of a hexaphosphate nucleotidehaving an energy transfer acceptor moiety, so as to incorporate thehexaphosphate nucleotide into the nucleic acid molecule thereby locatingthe polymerase and nucleotide in close proximity with each other togenerate the energy transfer signal; wherein the polymerase is a B103polymerase comprising SEQ ID NO:1, 2 or
 3. 3. A method for generating anenergy transfer signal comprising the steps of: contacting (i) apolymerase having altered nucleotide incorporation kinetics and linkedto an energy transfer donor moiety with (ii) a target nucleic acidmolecule which is base-paired with a polymerization initiation sitehaving a terminal 3′ OH group and with (iii) at least one type of anucleotide having an energy transfer acceptor moiety, so as toincorporate the nucleotide onto the terminal 3′ OH group therebylocating the polymerase and nucleotide in close proximity with eachother to generate the energy transfer signal; wherein the polymerase isa B103 polymerase comprising SEQ ID NO:1, 2 or
 3. 4. The method of claim1, further comprising the steps of: a) exciting the energy transferdonor moiety with an excitation source; and b) detecting the energytransfer signal from the energy transfer donor moiety and the energytransfer acceptor moiety that are in close proximity to each other. 5.The method of claim 1, further comprising the steps of: a) exciting theenergy transfer donor moiety with an excitation source; b) detecting theenergy transfer signal from the energy transfer donor moiety and theenergy transfer acceptor moiety which are in close proximity to eachother; and c) identifying the energy transfer signal from the energytransfer accepter moiety.
 6. The method of claim 1, wherein the alterednucleotide incorporation kinetics includes altered polymerase binding tothe target molecule, altered polymerase binding to the nucleotide,altered polymerase catalyzing nucleotide incorporation, altered thepolymerase cleaving the phosphate group or substituted phosphate group,and/or altered polymerase releasing the cleavage product.
 7. The methodof claim 1, wherein the energy transfer donor moiety is a nanoparticleor a fluorescent dye.
 8. The method of claim 7, wherein the nanoparticleis an inorganic fluorescent nanoparticle.
 9. The method of claim 7,wherein the nanoparticle is 1-20 nm in its largest dimension.
 10. Themethod of claim 7, wherein the nanoparticle is a non-blinkingnanoparticle.
 11. The method of claim 1, wherein the nucleic acidmolecule is a DNA molecule.
 12. The method of claim 1, wherein thenucleic acid molecule is immobilized to a surface.
 13. The method ofclaim 1, wherein the at least one type of nucleotide comprises 3-10phosphate groups.
 14. The method of claim 1, wherein a terminalphosphate group of the at least one type of nucleotide is linked to theacceptor moiety.
 15. The method of claim 1, wherein the at least onetype of nucleotide is adenosine, guanosine, cytosine, thymidine oruridine.
 16. The method of claim 1, wherein the polymerase is contactedwith at least two types of nucleotide.
 17. The method of claim 16,wherein the at least two types of nucleotides are each linked to adifferent type of energy transfer acceptor moiety.
 18. The method ofclaim 17, wherein the energy transfer acceptor moiety is a fluorescentdye.